Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 01/07 Ludwig-Maximilians-Universität München

The goal of this study was to investigate whether there is a “critical period” in the fourth and fifth week of life of laboratory rats, where an effect comparable to a socialisation to people can be achieved.

Preliminary tests on 6 female wistar-rats at the age of six weeks have been carried out to test different methods of non-invasive stress assessment in rats, and to develop a test procedure for the main experiments.
Not all investigated methods of stress assessment were applicable for the performed study. For example it could be shown that the determination of corticosterone metabolites in the faeces pointed out a distinct circadian rhythm, but no stress-related peak after performing the planned test procedure. The determination of IgA in the faeces has not been adequate to show stress-induced changes either.
The developed test procedure consisted of different behaviour tests: a modified home cage emergence test, a modified open field test, and a handtest. Additionally, a neck grip and a body-surface thermometry have been carried out, and catching has been evaluated.
In main experiment I, the animals have been allocated to different cages, splitting into experimental and control groups in a genetically balanced way. The rats have been housed in groups of three under standard conditions in type-IV Makrolon™ cages.
24 female wistar rats of age 21 days have been used for main experiment I (“early gentling”). The animals of the experimental group have been gentled for 10 minutes each day in the fourth and fifth week of life. All animals underwent tests to assess their behaviour towards people at the beginning of the sixth, eighth, tenth and fourteenth week of life, as well as at the age of six, six and a half and nine months. Following the tests at the beginning of the fourteenth week of life and at the age of six months the same test procedure has been carried out by a person unfamiliar to the rats (test with unfamiliar person).
12 female wistar rats, which were sisters of the animals in main experiment I and had been purchased together with them at the age of 21 days, have been used for main experiment II (“late gentling”). All animals were tested for the first time at the age of 6 months. In this test, as in the subsequent test with the unfamiliar person, they served as the zero check for the animals of main experiment I. Following these tests, the animals were allocated into experimental and control groups. The six animals of the experimental group were gentled for 10 minutes each day for two weeks. After the two weeks of gentling, at the age of six and a half months, and at the age of nine months, the rats of both groups have been tested to assess their behaviour towards people.
For the main experiment III (early “intensified gentling”) 24 female wistar rats of the age of 21 days have been used. The animals of the experimental group were gentled in the fourth and fifth week of life for 10 minutes twice each day. Furthermore, the animals were talked to during the gentling and recieved a food reward. The tests have been performed analogously to the tests of main experiment I.

All different parts of the test procedure have been analysed separately. Furthermore five principal target figures (PTF), summarising some results, have been defined. PTF 1 summarised the audible vocalisation and the freezing behaviour during the catching tests, PTF 2 the audible vocalisation and biting during the manipulations and the neck grip as well as the abortion of the neck grip, PTF 3 the behaviour during the handtest, PTF 4 the behaviour towards the stressor in the open field and PTF 5 summarised catching from the open field, without vocalisation. High values have been rated as “tameness”.

The “early gentling” of main experiment I had a distinct effect on the behaviour towards people. The animals of the experimental group showed a significantly (p0.05) higher tameness” than the animals of the control group i

Ziel der vorliegenden Arbeit ist es, einen Überblick über biologische Rhythmen bei den Nutztieren Pferd, Rind, Schwein, Legehennen und Enten zu erstellen. Weiterhin soll dargestellt werden, inwieweit die dem Tier eigenen biologischen Rhythmen unter den derzeit üblichen Haltungsbedingungen ungestört ablaufen können, und welche Änderungen der Haltungs- und Managementbedingungen gegebenenfalls geeignet wären, den natürlichen Ablauf dieser Rhythmen zu fördern.

Das Fortpflanzungsgeschehen zeigt bei allen im Rahmen dieser Literaturstudie berücksichtigten Tierarten einen circannualen Rhythmus, dessen primärer Zeitgeber die Photoperiode ist. Während der jährliche Rhythmus der Reproduktionsaktivität beim Pferd, bei der Legehenne und bei der Ente vielfach beschrieben wird, zählen das Rind und das Schwein allgemein zu den Tierarten mit ganzjährigem Reproduktionsgeschehen. Obwohl domestizierte Rinder und Schweine grundsätzlich die Möglichkeit zur ganzjährigen Fortpflanzung besitzen, deutet sich auch bei diesen Tierarten ein zugrunde liegender circannualer Rhythmus der Reproduktionsaktivität an.
Das Wach-Schlafverhalten landwirtschaftlicher Nutztiere unterliegt dem Einfluss circadianer und ultradianer biologischer Rhythmen. Der Ablauf dieser Rhythmen ist dabei für jede Tierart charakteristisch. Die Photoperiode ist als wichtiger Zeitgeber für den Wach-Schlafrhythmus anzusprechen.
Circadiane und ultradiane Rhythmen prägen die motorische Aktivität der Tierarten Pferd, Rind, Schwein, Legehennen und Enten, wobei auch hier der Verlauf der Rhythmen einem tierartspezifischen Muster folgt. Der gestörte Ablauf des biologischen Rhythmus der motorischen Aktivität, dessen primärer Zeitgeber die Photoperiode ist, wird in Zusammenhang mit dem Auftreten von Stereotypien gebracht.
Die Futteraufnahme unterliegt einem tierartspezifischen circadianen Rhythmus, der neben der Photoperiode von der Verfügbarkeit und Beschaffenheit der Nahrung und von sozialen Kontakten beeinflusst wird. Daneben kann die Futteraufnahme selbst als Zeitgeber für den Ablauf anderer Rhythmen dienen.
Das Sozial- und Komfortverhalten spielt bei den in größeren Gemeinschaften lebenden Nutztieren Pferd, Rind, Schwein, Legehennen und Enten eine wichtige Rolle. Der Einfluss circadianer Rhythmen deutet sich auch bei diesen Verhaltensweisen, die unter anderem für das Wohlbefinden der Tiere von Bedeutung sind, an.
Der Verlauf der Körpertemperatur folgt beim landwirtschaftlichen Nutztier einem circadianen Rhythmus, der allerdings nicht bei allen Tierarten gleich stark ausgeprägt ist. Der Rhythmus der Körpertemperatur ist endogenen Ursprungs, wird jedoch durch andere Rhythmen, wie den Rhythmus der motorischen Aktivität, den Wach-Schlafrhythmus und den Futteraufnahmerhythmus, beeinflusst.
Circannuale, circadiane und ultradiane Rhythmen prägen den Verlauf der Corticosteroidkonzentration. Die biologischen Rhythmen unterliegen dem Einfluss der Photoperiode, des Reproduktionsgeschehens und des Alters der Tiere. Der Verlauf des circadianen Rhythmus der Glucocorticoidsekretion kann möglicherweise zur Beurteilung der Haltungsumwelt, insbesondere zum Nachweis von chronischem Stress, herangezogen werden.
Der Verlauf einer Reihe weiterer Hormone wird beim landwirtschaftlichen Nutztier durch endogene Steuerungsvorgänge beeinflusst. So unterliegen die Konzentrationen der Schilddrüsenhormone T3 und T4 und einiger Sexualhormone, sowie die Konzentration des Hormons Prolactin, endogenen Rhythmen.
Die Funktion des Immunsystems folgt einem circannualen Rhythmus, dessen primärer Zeitgeber die Photoperiode ist. Daneben werden circannuale und circadiane Rhythmen hämatologischer und biochemischer Parameter beobachtet.

Obwohl sich eine Vielzahl von Studien mit dem Verlauf bestimmter biologischer Rhythmen beim Nutztier beschäftigt, fehlen Studien unter kontrollierten und standardisierten Bedingungen zu chronobiologischen Fragestellungen beim Nutztier weitgehend. D

Orally and intravenously administered glucose yields comparable levels of glucose in plasma but different responses of insulin-release. Specialised mucosa cells react to glucose contents in chyme with release of glucagon-like peptide-1 (GLP-1) into the portal bloodstream, a peptide hormone, that makes beta-cells more sensitive to the subsequent glucose stimulus and thereby enhances the insulin-release exclusively under increased blood glucose levels without causing the insulin-release directly. The capacity of this entero-insular axis is limited primarily by the endogenous enzyme dipeptidyl peptidase IV (DPP-IV).
Prevailingly the most progressive approach to therapy of non-insulin-dependent diabetes mellitus (NIDDM) in human medicine is targeted on the inhibition of DPP-IV, in order to raise GLP-1 concentration in plasma and to increase consequently the insulin-release in dependence on elevated blood glucose levels. In the limelight of discussion are the advantages of certain avoidance of insulin-caused hypoglycaemia on the one hand and the possibility of oral administration of the antienzyme on the other hand.
In contrast to the condition in dogs, where the disease manifests almost exclusively in the form of secondary (insulin-dependent) diabetes mellitus, the type 2 (NIDDM) predominates in humans as well as in cats, whence it comes that the employment of oral antidiabetics in the latter species is quite promising, as the experience with the sulfonyl urea derivative glipizide has shown. As however the residual capacity of beta-cells of feline diabetics with regard to the release of endogenous insulin is subject to considerable variation, the risk of inducing hypoglycaemia is given by use of both insulin and sulfonyl urea derivatives, whereas this threat is circumvented by use of DPP-IV-inhibitors.
In the present thesis the effect of DPP-IV inhibition on the elimination kinetics of GLP-1 is described by means of systematically combined infusion of the enzyme’s substrate (GLP-1) and its antienzyme in the rat model. The maximum effective dose of DPP-IV inhibitor is determined and the portion of hepatic clearance and whole blood clearance of total elimination of GLP-1 without concurrent DPP-IV inhibition is quantified approximately by means of isolated perfused rat livers and in-vitro-experiments. Finally the dependence of results on the respective experimental design is demonstrated.
The total clearance of GLP-1 as substrate of DPP-IV without concurrent inhibition amounts to at least 57 ± 17 (mL/min)/kg and under influence of the DPP-IV-inhibitor to at least 22 ± 2 (mL/min)/kg. The value of clearance is highly dependent on the respective substrate concentration in plasma, so that the values range from 139 ± 57 (mL/min)/kg to 73 ± 15 (mL/min)/kg within the bounds of physiological GLP-1 concentrations in plasma. The maximum increase of GLP-1 levels in plasma (factor 3.4) on average of all GLP-1 infusion rates was attained by a DPP-IV inhibitor concentration in plasma of 0.4 µmol/L. The liver as central detoxication-organ figures out at 57 % (34/60) of total clearance, the soluble DPP-IV fraction in plasma however accounts for only about 1 % (0.7/60) of total clearance. The relatively low hepatic extraction ratio of 30 ± 11 % reversely allows seven out of ten GLP-1 molecules to enter the systemic circulation in spite of the first-pass-effect and thereby to reach possibly the receptors on the beta-cells (70 % bioavailability). The half-life of GLP-1 (7-36 amide) in the isolated perfused rat liver amounts to 5.0 ± 1.3 minutes, hereby differing statistically not significantly (p = 0.114) from the half-life of GLP-1 (7-37) with 6.1 ± 1.6 minutes.

Long-term effects of Dexmedetomidin on the expression of apoptosis-regulating proteins after incomplete cerebral ischemia in rats

This study investigates the effect of the α2-adrenozeptor agonist Dexmedetomidine on the expression of the apoptosis-regulating proteins Bax, p53, Bcl-2 and Mdm-2 following incomplete cerebral ischemia in rats within a period of 28 days from the insult. 72 fasted male Sprague-Dawley rats (400 g) were randomly assigned to one of the following groups:
Group 1: (n = 32, controls): fentanyl and N2O/O2 (FiO2 = 0.33)
Group 2: (n = 32, dexmedetomidine) : fentanyl and N2O/O2 (FiO2 = 0.33), administration of dexmedetomidine intraperitoneal 30 min before ischemia,
the animals of these groups were randomly assigned in groups (n = 8) with a postischemic observation period of 1, 3, 7 or 28 days.
Group 3: (n = 8, naive): without treatment, show reference value
The apoptosis-regulating proteins Bax, p53, Bcl-2 and Mdm-2 in the hippocampal regions were analysed qualitatively and quantitatively by using the Immunfluorescence-technique and the Western-Blot-technique. The concentration of the pro-apoptotic protein Bax is decreased in the hippocampus for at least 28 days after cerebral ischemia. The anti-apototic Bcl-2 protein was increased during the first three days after cerebral ischemia in group 2 compared to group 1. The Mdm-2 protein of group 2 was significantly increased during the whole period of investigation in the Western-Blot-analysis.

In a retrospective study including the records of 628 patients of the Medizinische Kleintierklinik München, the significance of electromyography (EMG), electroneurography (ENG) and auditory evoked potentials (AEP) for the clinical diagnosis was determined.
Indications for AEP examination were screening for hereditary deafness (59.5%), evaluation of hearing abilities for other reasons (19.2%), vestibular syndrome (13.4%), suspicion on brainstem disease (2.3%), suspicion on hereditary ataxia in the Jack Russell terrier (1.8%) and facial paralysis (3.8%).
Indication for the EMG/ENG examination were exclusion of (4.0%) or suspicion on (10.1%) lumbosacral syndrome, lameness of unknown origin (23.5%), paraparesis (8.7%) and tetraparesis (3.4%) of unknown origin, clinical suspicion on a focal neuropathy (15.1%), on a generalized neuromuscular disorder (30.5%), or on masticatory myositis (3.4%), and miscellaneous (1.3%).
The electrodiagnostic examination was of decisive significance for the diagnosis of the following diseases: congenital deafness, acquired deafness, focal neuropathies.
The electrodiagnostic examination was mostly not decisive, but characterized the disease and indicated the final clinical diagnosis in following indications: (1) AEP for vestibular syndrome, suspicion on brainstem disease, hereditary ataxia of the Jack Russell terrier and facial paresis. (2) EMG and ENG for lameness of unkown origin, suspicion on lumbosacral syndrome, suspicion on a generalized neuromuscular disorder, suspicion on masticatory myositis.
For some neuromuscular diseases, sensitivity (S%) and positive predictive value (PPV%) of the electrodiagnostic examination were established:
In lumbosacral syndrome, S% was 87.5% and PPV% was 66.7%. In focal neuropathy, S% was 98.3% and PPV% was 100%. In generalized neuromuscular disorders, S% was 91.6% and PPV% was 85.3%; within this group, polymyopathies were diagnosed with S% = 63.6%, polyneuropathies with S% = 90.9%, and neuro-muscular transmission disorders with S% = 75 %.
Thus, multiple indications were shown, in which the electrodiagnostic examination contributed essentially to the clinical diagnosis.

The aim of this study was to examine if the assessments of sperm quality can be better objectified and standardized by using flow cytometric examinations. For this investigation temporary deterioration of sperm quality was induced by a local heating of testes in bulls.

The scrotal surface temperature of 4 bulls was increased during the local heating of testis by enclosing the entire scrotum for 48 hours by 6.4°C to 7.1°C. Semen was collected 3 times a week from Day -7 to Day 61 and once a week from Day 68 to Day 83 (Day 0 = the day of scrotal insulation). Each ejaculate was conventionally examined (numbers of total spermatozoa, sperm concentration, morphology and proportion of vital spermatozoa after staining with bromphenol blue nigrosin). The percentage of motile spermatozoa and progressive motile spermatozoa of motile sperm were determined with a computer-assisted motility analysis. Using flow cytometry the proportions of sperm with the following parameters were examined and analyzed: intact plasma membranes after staining with SYBR®14/PI, high mitochondrial membrane potentials after staining with JC1, damaged acrosome status after staining with FITC-PNA/SYTO®17/PI, and sperm with defective chromatin structure or rather with high DNA fragmentation index (DFI) by SCSA.

The deterioration of sperm quality after elevating testicular temperature was correspondent to a large extent to the typical changes of the conventional sperm parameters, which result is in agreement with previous similar studies. After testicular hyperthermia (Day 0) changes of sperm quality occurred in the following sequence. There was a notable increase in secondary sperm abnormalities as well as a decrease in sperm motility on Day 9 after testicular hyperthermia. The proportions of vital spermatozoa after staining with bromphenol blue nigrosin, total sperm count and sperm concentration were decreased on Day 12 for the first time. At the same time the primary sperm abnormalities began to increase. The primary abnormalities most frequently encountered were morphological head defects of spermatozoa.

Concurrent with the changes specified above the following alterations of sperm parameters were observed with flow cytometry. Beginning on Day 7 after testicular hyperthermia sperm with defective acrosome status increased. From Day 9 on sperm with intact plasma membranes and sperm with high mitochondrial membrane potentials began to decrease. On Day 12 after testicular hyperthermia the proportion of sperm with defective chromatin structure (spermatozoa with high DFI) started to increase significantly.

The relationships between the proportions of defective sperm chromatin structure assessed with the SCSATM test and the proportions of sperm head defects were highly significant (r = 0.81; P 0.0001). The proportion of sperm with a high mitochondrial membrane potential correlated positively to the sperm motility (r = 0.83; P 0.0001). Significant correlations between the viability assessed by light microscopy and the percentages of spermatozoa with intact plasma membrane obtained by flow cytometry (r = 0.77; P 0.0001) occurred.

The increase of the proportion of the sperm with defective chromatin structure (spermatozoa with high DFI) was more clearly pronounced than that of the morphological abnormal sperm heads. This result indicates that heat stress had led to chromosome defects not only in morphologically abnormal but also in normally appearing sperm cells. In addition, morphological sperm abnormalities, especially sperm head defects, may be indicative of chromosome abnormalities also in normally appearing sperm cells of an ejaculate.

This study shows that flow cytometric assessments of the ejaculate is a reliable and objective method in semen investigation. It provides additional important information about the sperm quality. In further studies, it has to be clarified, whether the prospective fertilization capacity from an ejaculat