Age- and Sex-Associated Effects on Acute-Phase Proteins in G?ttingen Minipigs

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40–48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli. Abbreviations: APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; HFD, high-fat diet; HFD+D, high fat diet + diabetes; PAGP, porcine α1 acid glycoprotein; PMAP, pig major acute-phase protein; SAA, serum amyloid AInflammation is involved in a number of important and increasingly widespread human diseases, including inflammatory bowel diseases, cancers, infections, metabolic diseases like obesity and diabetes, and cardiovascular diseases like atherosclerosis.^{1,5,7,11,20,41} The systemic response to inflammation is the acute-phase response (APR) which, together with innate immune responses, prevents infection, clears pathogens, and contributes to inflammation resolution and the healing process. The APR has been extensively described in humans^10,22 and other mammals,^8,14,29,31 and in all cases, it is regulated by cytokines including IL6 and TNFα.^21,30 The APR is activated by many different stimuli, including trauma, infection, stress, neoplasia, and inflammatory stimuli, resulting in significant changes in the circulating concentrations of the so-called acute-phase proteins (APP). The APP are synthetized primarily by the liver and can be divided into positive and negative APP depending on whether their concentration in plasma increases (positive) or decreases (negative) in response to a stimulus.¹⁰ In addition, they can be divided into major and minor APP, depending on the magnitude of their concentration change after a given stimulus.²² Because the concentrations of the APP change in response to a given stimulus, their serum or plasma levels can be used diagnostically as biomarkers of disease severity and progression or to evaluate the effect of various interventions.^8,14,31 The APP show different kinetics after a stimulus, with C-reactive protein (CRP) and serum amyloid A (SAA) displaying rapid increases and normalization after the stimulus has been removed, whereas haptoglobin shows a later and more prolonged response.^10,31 The APR may be transient and revert to normal with recovery, or it can persist, as during chronic conditions.²¹ Importantly, APP and their kinetics differ somewhat between species.³¹To further elucidate the involvement of inflammation in human diseases, accurate animal models of inflammation, including species validated biomarkers of inflammation, are needed. Mouse models are commonly used in many research areas, but their response to several different inflammatory conditions is not comparable to that of humans, and therefore the predictive validity of these models may be limited.³⁹ Pigs are highly comparable to humans with respect to anatomy and physiology,⁴⁴ and their APR to various stimuli has been described.^14,23,26 In general, the APR and the resulting changes in APP seem to be very similar in pigs compared with humans, with CRP, haptoglobin, and SAA being major positive APP and albumin being a negative APP.¹⁴ In humans, α1-acid glycoprotein (AGP) is a positive APP but has been reported to either increase,¹⁷ remain unchanged^23,45 or to decrease¹² in pigs, depending on the stimulus investigated. The concentrations of some of the major APP characterized in domestic pigs show significant effects of age and sex.^32,34 In addition to age and sex effects, significant differences in APP between herds have been observed, most likely reflecting different pathogenic pressures in the different herds.³² Furthermore, some indications exist for possible interbreed differences in APP concentrations, although this possibility has not been investigated in detail.¹²Minipigs are especially relevant in biomedical research, given their smaller size and well-defined microbiology and genetics.⁴ Göttingen minipigs are a useful model for several conditions involving inflammation and the APR, including infection,² obesity,¹⁹ diabetes²⁴ and atherosclerosis,¹⁸ and different APP have already been used as biomarkers in some of these models.² Therefore, existing data suggest that APP commonly applied in human medicine could be relevant in Göttingen minipigs as well. However, the APR and reference values of APP, including the potential influence of age and sex indicated in other studies, have not been investigated systematically in this breed.^12,32,34The objective of the current study was to establish reference values of selected APP in normal Göttingen minipigs, including evaluation of the possible effects of age and sex. In addition, the effects of housing condition and acute and chronic inflammatory stimuli were assessed.     

Characterisation of Gut Microbiota in Ossabaw and G?ttingen Minipigs as Models of Obesity and Metabolic Syndrome

Background

Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs.

Methods and Findings

The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs’ cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine.

Conclusion

Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.     

High Fat,Low Carbohydrate Diet Limit Fear and Aggression in G?ttingen Minipigs

High fat, low carbohydrate diets have become popular, as short-term studies show that such diets are effective for reducing body weight, and lowering the risk of diabetes and cardiovascular disease. There is growing evidence from both humans and other animals that diet affects behaviour and intake of fat has been linked, positively and negatively, with traits such as exploration, social interaction, anxiety and fear. Animal models with high translational value can help provide relevant and important information in elucidating potential effects of high fat, low carbohydrate diets on human behaviour. Twenty four young, male Göttingen minipigs were fed either a high fat/cholesterol, low carbohydrate diet or a low fat, high carbohydrate/sucrose diet in contrast to a standard low fat, high carbohydrate minipig diet. Spontaneous behaviour was observed through video recordings of home pens and test-related behaviours were recorded during tests involving animal-human contact and reaction towards a novel object. We showed that the minipigs fed a high fat/cholesterol, low carbohydrate diet were less aggressive, showed more non-agonistic social contact and had fewer and less severe skin lesions and were less fearful of a novel object than minipigs fed low fat, high carbohydrate diets. These results found in a porcine model could have important implications for general health and wellbeing of humans and show the potential for using dietary manipulations to reduce aggression in human society.     

Gastrointestinal Acute Radiation Syndrome in G?ttingen Minipigs (Sus Scrofa Domestica)

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; ⁶⁰Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.Abbreviations: GI-ARS, gastrointestinal acute radiation syndromeNo drugs have been approved for the use of preventing gastrointestinal acute radiation syndrome (GI-ARS) in irradiated patients. The effect of the acute exposure of the GI tract to radiation traditionally has been associated with the inhibition of mitotic activity in intestinal crypts and the interrupted migration of GI epithelial cells from the crypts to the tips of the villi, leading to denudation of the intestinal mucosal barrier. The monolayer of epithelial cells lining the GI mucosa performs many functions of vital importance, including electrolyte transport, secretion of digestive fluids, absorption of nutrients, excretion of toxins, and providing a barrier to the luminal environment and commensal bacteria. The loss of intestinal barrier integrity results in the loss of nutrients, water, and electrolytes; increased permeability to bacteria and antigens; sepsis; inflammation; and organ dysfunction.In humans, GI-ARS usually manifests at doses exceeding 5 to 6 Gy;¹⁰ death occurs within 2 wk in the absence of treatment. Radiation-induced GI damage is accompanied by bone marrow suppression; the sequelae of GI-ARS and hematopoietic ARS partially overlap but do not necessarily develop concomitantly.²³ Characteristic of GI-ARS is the early onset of symptoms (nausea, vomiting, watery diarrhea, cramps) within a few hours, and the overt illness is marked by vomiting and diarrhea. Bacterial translocation from the intestinal tract, loss of crypts, shortening of villi, decreased citrulline levels, onset of abdominal pain, vomiting, and diarrhea are accepted markers of the GI syndrome.²² Cardiovascular collapse, fluctuations in electrolyte concentrations, severe hemorrhage, and sepsis likely contribute to acute renal and multiorgan failure.²³Well-characterized animal models are required for efficacy testing of radiation countermeasures. So far, NHP are the only large animal model well-characterized in regard to the dose–survival relationship, symptoms, vital signs, and GI histology of GI-ARS.¹¹ In NHP, the LD_50/15 for GI-ARS in the presence of medical management including blood product transfusion is estimated to be 11.33 Gy.¹¹ Animals in the cited study¹⁰ were characterized by shorter survival time compared with hematopoietic ARS, diarrhea, dehydration, and dose-dependent loss of body weight, intestinal crypts, and villi. In addition to the total body irradiation model, a partial-body irradiation model was established in NHP, by using 5% sparing of the bone marrow, to evaluate the long-term effects of radiation on the GI system and to study concomitant subsyndromes and organ injuries, including bone marrow and lungs.¹⁰ Advantages of the partial body irradiation model are the ability to assess the development of various subsyndromes over time and the development of a polypharmacy approach targeting multiple organs. In dogs, acute GI-ARS was produced consistently by a single dose of 9.38 Gy to the abdomen.²⁰ In rodents, GI damage was induced by 8 to 15 Gy total body irradiation, depending upon the species and strain,^5,6,17,18 and was characterized by accelerated death, poor nutrient absorption, crypt depletion, intestinal epithelial cells denudation, infections, diarrhea, dehydration, and weight loss. In mice, both acute and delayed effects of the GI syndrome have been identified; study endpoints evaluating medical countermeasures against GI-ARS have been proposed.^2,3 Acute effects were assessed at several levels of bone-marrow shielding and supportive care and were demonstrated by the presence of diarrhea, apoptosis, villus blunting, crypt depletion, loss of mucosal integrity, and ulcerations in the GI tract. Delayed effects included higher-than-normal levels of apoptotic and mitotic crypt cells, premature reductions in gut function, and the presence of adenomas.^2,3Additional large animal models are urgently needed to expedite drug testing and marketing under FDA regulations. Minipigs may be a potential alternative model for studying hematopoietic ARS,¹⁵ but their high sensitivity to radiation prompts doubt regarding the feasibility of GI-ARS studies in this model. In the current study, we filled an important gap in development of minipigs as a model for ARS and addressed potential concerns regarding the feasibility of inducing classic GI-ARS in minipigs before they succumb to cardiovascular or respiratory complications. In previous publications, we documented hematopoietic ARS and accelerated hematopoietic ARS at higher radiation doses.^14,15 However, despite sporadic reports of specific types of radiation-induced injury to the GI tract of swine after total body irradiation, GI-ARS in that species has not been addressed comprehensively. To this end, we used a population of minipigs that was completely different from that in the previous studies and exposed the animals to much higher radiation doses than had been used previously. The subsequent results were markedly different from those of the previous reports, including loss of GI crypts and large decreases in circulating citrulline, both of which are important signs of GI-ARS. Our results demonstrate convincingly that GI-ARS occurs in swine and place this subsyndrome in the context of others studied comprehensively in the same strain. This achievement is an important milestone in the development of this much-needed large animal model of ARS, which promises to provide an alternative to NHP and dogs.We show here that exposure of minipigs to γ irradiation in the range 5 to 12 Gy resulted in the development of signs typical of GI-ARS, and we discuss the importance of choosing the appropriate endpoint criteria for the different subsyndromes of ARS. Most minipigs died within 10 d, shifting the question from the feasibility of inducing GI-ARS in minipigs to the levels of supportive care appropriate for drug efficacy testing in this model.     

Cognitive Performance of G?ttingen Minipigs Is Affected by Diet in a Spatial Hole-Board Discrimination Test

Consumption of a high energy diet, containing high amounts of saturated fat and refined sugar has been associated with impairment of cognitive function in rodents and humans. We sought to contrast the effect of a high fat/cholesterol, low carbohydrate diet and a low fat, high carbohydrate/sucrose diet, relative to a standard low fat, high carbohydrate minipig diet on spatial cognition with regards to working memory and reference memory in 24 male Göttingen minipigs performing in a spatial hole-board discrimination test. We found that both working memory and reference memory were impaired by both diets relative to a standard minipig diet high in carbohydrate, low in fat and sugar. The different diets did not impact levels of brain-derived neurotrophic factor in brain tissue and neither did they affect circulatory inflammation measured by concentrations of C-reactive protein and haptoglobin in serum. However, higher levels of triglycerides were observed for minipigs fed the diets with high fat/cholesterol, low carbohydrate and low fat, high carbohydrate/sucrose compared to minipigs fed a standard minipig diet. This might explain the observed impairments in spatial cognition. These findings suggest that high dietary intake of both fat and sugar may impair spatial cognition which could be relevant for mental functioning in humans.     

Regression of diet-induced atherosclerosis in G?ttingen miniature swine

The regression of diet-induced atherosclerosis in G?ttingen Miniature Swine was investigated after a 6-month induction period. At 1 month after feeding a high-cholesterol and high-fat diet, levels of beta-lipoprotein, total cholesterol, free fatty acids and phospholipid had increased rapidly and the high levels were maintained throughout the 6 month induction period. Morphological features at 6 months showed fatty streaks in the thoracic aorta and fibrous plaques in the abdominal aorta. After return to the conventional diet at 6 months, serum lipids decreased rapidly and maintained the baseline level throughout the 9 month regression period. Histopathological findings showed the regression of fatty streaks but the fibrous plaques did not regress. The present study therefore confirms the regression of fatty streaks in the aorta of G?ttingen Miniature Swine by the administration of a cholesterol lowering diet.     

Novel Technique for Retroperitoneal Implantation of Telemetry Transmitters for Physiologic Monitoring in G?ttingen Minipigs (Sus scrofa domesticus)

Telemetric monitoring of physiologic parameters in animal models is a critical component of chemical and biologic agent studies. The long-term collection of neurobehavioral and other physiologic data can require larger telemetry devices. Furthermore, such devices must be implanted in a location that is safe, well-tolerated, and functional. Göttingen minipigs (Sus scrofa domesticus) present an ideal large animal model for chemical agent studies due to their relatively small size, characterized health status, and ease of training and handling. We report an effective approach to implanting a novel device to measure transthoracic impedance to approximate respiratory tidal volume and rate in Suidae. We tested the approach using 24 male Göttingen minipigs. A ventral midline abdominal incision extending from the umbilicus to the prepuce was followed by a paramedian incision of the parietal peritoneum and dorsal blunt dissection to create a retroperitoneal pocket. The device was anchored inside the pocket to the internal abdominal musculature with 3-0 nonabsorbable suture, biopotential leads were routed through the abdominal musculature, and the pocket was closed with 3-0 absorbable suture. Paired biopotential leads were anchored intermuscularly at the level of the seventh rib midway between spine and sternum bilaterally to provide surrogate data for respiratory function. Postoperative recovery and gross pathology findings at necropsy were used to assess safety and refine the surgical procedure. Results demonstrated that this procedure permitted effective monitoring of complex physiologic data, including transthoracic impedance, without negatively affecting the health and behavior of the animals.Telemetric monitoring of physiologic parameters in animal models is a critical component of chemical⁹ and biologic agent studies.^32,36 The attenuation of the adverse effects of these agents by using medical countermeasures can be assessed also.^21,25,28,35 The long-term collection of neurobehavioral and other physiologic data can require large telemetry devices. Furthermore, such devices must be implanted in a location that is safe, well-tolerated, and functional. Potential complications of inserting telemetry devices include infection,²⁹ dehiscence,⁷ neoplasia,³³ and extrusion.²⁰Göttingen minipigs (Sus scrofa domesticus) present an ideal large animal model for chemical agent studies due to their relatively small size, characterized health status, and ease of training and handling, including for physiologic studies.^12,15,16,30 The lightly pigmented skin, sparse haircoat, fine intersecting lines of epidermal sulci, lipid biophysical properties, and epidermal turnover kinetics of Göttingen minipigs are similar to those in humans.^22,34 Swine in general are a particularly relevant animal model for assessing percutaneous absorption exposure to chemical agents.^8,11 Percutaneous application of agent represents the most likely real-world threat scenario for persistent chemical agents.^8,11 Extensive in vivo and in vitro studies have characterized percutaneous absorption,^10,13,31 protection,⁵ and decontamination²³ in swine models.Previously, respiratory impedance has been measured by using implantable telemetry devices in nonhuman primates,² and respiratory parameters have been determined by using external impedance-based technology in minipigs¹ and dogs.²⁴ We report an effective approach to implanting a device for measuring transthoracic impedance to approximate respiratory tidal volume and rate in minipigs.     

Osteoarthritis in the Knee Joints of G?ttingen Minipigs after Resection of the Anterior Cruciate Ligament? Missing Correlation of MRI,Gene and Protein Expression with Histological Scoring

IntroductionThe Göttingen Minipig (GM) is used as large animal model in articular cartilage research. The aim of the study was to introduce osteoarthritis (OA) in the GM by resecting the anterior cruciate ligament (ACLR) according to Pond and Nuki, verified by histological and magnetic resonance imaging (MRI) scoring as well as analysis of gene and protein expression.

Materials and Methods

The eight included skeletally mature female GM were assessed after ACLR in the left and a sham operation in the right knee, which served as control. 26 weeks after surgery the knee joints were scanned using a 3-Tesla high-field MR tomography unit with a 3 T CP Large Flex Coil. Standard proton-density weighted fat saturated sequences in coronal and sagittal direction with a slice thickness of 3 mm were used. The MRI scans were assessed by two radiologists according to a modified WORMS-score, the X-rays of the knee joints by two evaluators. Osteochondral plugs with a diameter of 4mm were taken for histological examination from either the main loading zone or the macroscopic most degenerated parts of the tibia plateau or condyle respectively. The histological sections were blinded and scored by three experts according to Little et al. Gene expression analysis was performed from surrounding cartilage. Expression of adamts4, adamts5, acan, col1A1, col2, il-1ß, mmp1, mmp3, mmp13, vegf was determined by qRT-PCR. Immunohistochemical staining (IH) of Col I and II was performed. IH was scored using a 4 point grading (0—no staining; 3-intense staining).

Results and Discussion

Similar signs of OA were evident both in ACLR and sham operated knee joints with the histological scoring result of the ACLR joints with 6.48 ± 5.67 points and the sham joints with 6.86 ± 5.84 points (p = 0.7953) The MRI scoring yielded 0.34 ± 0.89 points for the ACLR and 0.03 ± 0.17 for the sham knee joints. There was no correlation between the histological and MRI scores (r = 0.10021). The gene expression profiles as well as the immunohistochemical findings showed no significant differences between ACLR and sham knee joints. In conclusion, both knee joints showed histological signs of OA after 26 weeks irrespective of whether the ACL was resected or not. As MRI results did not match the histological findings, MRI was obviously unsuitable to diagnose the OA in GM. The analysis of the expression patterns of the 10 genes could not shed light on the question, whether sham operation also induced cartilage erosion or if the degeneration was spontaneous. The modified Pond-Nuki model may be used with reservation in the adult minipig to induce an isolated osteoarthritis.     

Diurnal variation and age-related changes of bone turnover markers in female Göttingen minipigs

We investigated diurnal variation and age-related changes in bone turnover markers in female Gottingen minipigs. Ten females, 6-9 months of age, were used for confirmation of diurnal variation. Blood was collected at 3 h intervals for 24 h, and bone-specific alkaline phosphatase and intact osteocalcin (OC) levels were determined by enzyme immunoassay and radioimmunoassay, respectively. Urine was collected at 3 h intervals for 24 h using a tray attached to the bottom of the cage. The levels of N-terminal telopeptide of type I collagen (NTX) were determined by enzyme immunoassay. Pyridinoline and deoxypyridinoline were measured by high performance liquid chromatography. OC and NTX exhibited diurnal variation (Kruskal-Wallis test, P < 0.05), with the highest and lowest levels at 18:00 h (76.7 +/- 26.2 ng/ml) and 06:00 h (44.3 +/- 10.3 ng/ml), and at 03:00-05:59 h (550.4 +/- 82.4 nmol/micromol Cr) and 12:00-14:59 h (297.8 +/- 152.5 nmol/micromol Cr), respectively. In the study of age-related changes, blood and urine samples from 66 females (age range, 3-76 months) were examined to determine the bone turnover markers. All markers showed high correlations with age (0.569 < R(2) < 0.818). High levels of bone turnover markers were observed in young animals, decreasing with age (Kruskal-Wallis test, P < 0.01). The diurnal variation and age-related changes revealed in the present study will be useful in studies of bone diseases using female Gottingen minipigs.     

A linkage map of 243 DNA markers in an intercross of Göttingen miniature and Meishan pigs

A resource family of pigs has been constructed by using a boar of Göttingen miniature pig and two sows of Meishan pig as parents. In the construction of the family, two F1 males and 18 F1 females were intercrossed to generate 143 F2 offspring. The members of the family were genotyped using 243 genetic markers including 26 markers developed in our laboratory in order to generate a linkage map of markers for use in detecting quantitative trait loci (QTLs) in the family. The markers consisted of 237 microsatellites, five PRE-1 markers, and one RFLP marker. The linkage map was revealed to cover all 18 autosomes and the X chromosome; and the total length of the sex-averaged linkage map was calculated to be 2561 ·9 c m . Four out of the 26 markers developed in our laboratory ex-ended the current linkage map at the termini of chromosomes 1p, 5p, 11p, and Xq. The linkage maps of all the chromosomes except for chromosome 1 were found to be longer in females than in males. Concerning chromosome 1, the length of the linkage map showed no difference between females and males, which was attributed to low recombination rates between markers localized in the centromeric region in females. The average ratio of female-to-male recombination was calculated to be 1 ·55.     

Electrocardiographic monitoring in the Göttingen minipig

The purpose of the study reported here was to determine conditions for electrocardiographic monitoring in the G?ttingen minipig in view of its use as a second non-rodent species in toxicology studies. Electrocardiograms were recorded from conscious minipigs (6/sex) maintained in a sling. The three standard bipolar limb leads (I, II, III), the three augmented unipolar limb leads (aVR, aVL, aVF), the triangular Nehb-Sp?ri leads (dorsal, axial, ventral) and their corresponding unipolar leads were recorded, and automated analysis of amplitudes and intervals was made. Major QRS patterns were not observed for any of the bipolar and unipolar leads. For triangular leads, the amplitude of waves was higher than that for limb leads, and the rS pattern dominated for dorsal, axial ventral and aV(F)-Ventral leads. The qR pattern dominated in the aV(R)-dorsal lead, whereas consistency and dominant patterns were not observed for the aV(L)-axial lead. For limb leads, the position of the electrode affected the ECG. Electrodes placed on the cubital and stifle joints were the preferred positions since the P- and R-waves were clearly identifiable with amplitudes > 0.2 mV. Also, the T-wave amplitude was (positive or negative) > 0.2 mV in at least two leads, making the determination of the QT-interval accurate. For the triangular leads, the position of the electrode had less influence on the amplitude of deflections. However, if the axial lead is to be used for calculation of intervals and amplitudes, the xyphoid process is the preferred position. In conclusion, the triangular lead system is recommended for recording ECGs in minipigs. Limb leads could be used in connection. The cubital and stifle joints for standard limb leads and the neck, sacrum, and xyphoid process for triangular leads are the preferred positions for electrodes.     

Acquisition of visually guided conditional associative tasks in Göttingen minipigs

Fourteen Göttingen minipigs were trained on two different visually guided conditional associative tasks. In a spatial conditional task, a black stimulus signalled that a response to the left was correct, and a white stimulus signalled that a response to the right was correct. In a conditional go/no-go task, a blue stimulus signalled go, and a red stimulus signalled no-go. The pigs were trained until a behavioural criterion of 90% correct for each of two consecutive sessions. For the spatial conditional task, all pigs reached this criterion in 520 trials or less. For the conditional go/no-go task, all pigs, except three, reached this criterion in 1600 trials or less. Sows and boars learned equally fast. The tasks can be useful for the testing of cognitive function in pig models of human brain disorders.     

Measurements of insulin responses as predictive markers of pancreatic beta-cell mass in normal and beta-cell-reduced lean and obese Göttingen minipigs in vivo

At present, the best available estimators of beta-cell mass in humans are those based on measurement of insulin levels or appearance rates in the circulation. In several animal models, these estimators have been validated against beta-cell mass in lean animals. However, as many diabetic humans are obese, a correlation between in vivo tests and beta-cell mass must be evaluated over a range of body weights to include different levels of insulin sensitivity. For this purpose, obese (n = 10) and lean (n = 25) G?ttingen minipigs were studied. Beta-cell mass had been reduced (n = 16 lean, n = 5 obese) with a combination of nicotinamide (67 mg/kg) and streptozotocin (125 mg/kg), acute insulin response (AIR) to intravenous glucose and/or arginine was tested, pulsatile insulin secretion was evaluated by deconvolution (n = 30), and beta-cell mass was determined histologically. AIR to 0.3 (r(2) = 0.4502, P < 0.0001) or 0.6 g/kg glucose (r(2) = 0.6806, P < 0.0001), 67 mg/kg arginine (r(2) = 0.5730, P < 0.001), and maximum insulin concentration (r(2) = 0.7726, P < 0.0001) were all correlated to beta-cell mass when evaluated across study groups, and regression lines were not different between lean and obese groups except for AIR to 0.3 g/kg glucose. Baseline pulse mass was not significantly correlated to beta-cell mass across the study groups (r(2) = 0.1036, NS), whereas entrained pulse mass did show a correlation across groups (r(2) = 0.4049, P < 0.001). This study supports the use of in vivo tests of insulin responses to evaluate beta-cell mass over a range of body weights in the minipig. Extensive stimulation of insulin secretion by a combination of glucose and arginine seems to give the best correlation to beta-cell mass.     

Histological study on the arterial wall of G?ttingen miniature swine

In order to clarify the histological aspects of arterial walls of miniature swine, 41 arterial segments removed from each of 17 G?ttingen miniature swines/csk at 6-12 months after birth were examined by light microscopy. Every segment was classified into three types--elastic, transitional, and muscular--according to its histological architecture. Each type was identified on the basis of relative volume and disposition of elastic tissues, collagenous fibers, and smooth muscles in three coats, Tunica intima, Tunica media, Tunica externa. The distribution of each type was demonstrated in a transition from the elastic to the muscular type. The transitional type was designated as a representative type of artery in transitional regions between elastic and muscular types and had some characteristic structures like a mixture of both of them. This type also had longer regions in the back district with the heart as the starting point, such as the Aorta abdominalis, than that of the front. Every artery belonging to the three types had some different structures at different levels, respectively. It was supposed that the histological arrangement of tissues in the walls of a vessel from various parts of the arterial tree would respond properly to the functional demand.     

Discriminations, reversals, and extra-dimensional shifts in the Göttingen minipig

G?ttingen minipigs were trained on a set-shifting procedure involving discriminations, reversals, and extra-dimensional shifts. The discriminations used were black-white discriminations and right-left discriminations. The initial visual and spatial discrimination seemed equally difficult, and only for the visual modality was reversal found to be more difficult than the initial discrimination. Visual reversal was more difficult than spatial reversal, and a larger number of perseverative sessions were found for visual reversal compared to spatial reversal. The acquisition of the extra-dimensional shift from the visual to the spatial dimension was not inferior to the learning of spatial reversal. Neither was the acquisition of the extra-dimensional shift from the spatial to the visual dimension inferior to the learning of visual reversal. Thus, no evidence was found for attention to stimulus dimensions in discrimination learning of the pigs.     

Gender-associated differences in metabolic syndrome-related parameters in Göttingen minipigs

Gender-associated differences in pathophysiology and treatment of disease are an evolving area in human medicine that should be addressed in animal models. The aim of this study was to characterize gender differences in metabolic parameters of G?ttingen minipigs and to determine which gender has the metabolic profile that is most appropriate as a model for human metabolic syndrome. Blood samples were collected from fasted, lean male and female G?ttingen minipigs at 8 wk and 8 mo of age. Samples were analyzed for glucose, fructosamine, insulin, C-peptide, glucagon, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-c), free fatty acids, leptin, testosterone, and 17beta-estradiol. Insulin sensitivity and beta cell function were estimated by homeostasis model assessment and degree of obesity by measuring the abdominal circumference. Male minipigs had higher concentrations of both testosterone and estradiol. Female minipigs had a larger abdominal circumference and higher concentrations of C-peptide, insulin, triglyceride, total cholesterol, HDL-c and leptin but a lower concentration of free fatty acids and lower HDL-c:total cholesterol ratio. Compared with male minipigs, female minipigs were more insulin-resistant and had a higher beta-cell function. No gender-associated differences were found in any of the other investigated parameters. In conclusion, female minipigs were more obese and insulin-resistant and had a more atherogenic plasma profile than did their male counterparts and therefore may be better models for metabolic syndrome. Their high concentrations of both testosterone and estradiol may protect male minipigs from obesity and metabolic disturbances.     

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the terminal ileum of fistulated Göttingen minipigs

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with G?ttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10(6) to 10(7) per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.     

Parameters of glucose and lipid metabolism in the male Göttingen minipig: influence of age, body weight, and breeding family

The pig is useful as a model for human physiology and pathophysiology and could be an important supplement to the many available rodent models of diabetes mellitus. Due to their small size, G?ttingen minipigs are especially suitable for long-term studies. The aim of the study reported here was to establish reference values for a range of glucose and lipid homeostasis parameters of interest that could be used to identify possible diabetes-prone male G?ttingen minipig individuals, families, or age groups. Plasma samples from nonfed animals were analyzed for glucose, leptin, fructosamine, insulin, C-peptide, triglyceride, free fatty acids, and total cholesterol values. Breeding family had significant effects only on plasma triglyceride concentrations (P < 0.001). Plasma concentrations of glucose (P = 0.012), fructosamine (P < 0.001) and triglycerides (P < 0.001) increased significantly with age, whereas total cholesterol concentration decreased significantly (P = 0.001) with age. Age did not influence other parameters. In conclusion, glycemia and insulinemia increased with age and body weight, possibly indicating a small deterioration in insulin sensitivity with age. It is, therefore, hypothesized that older, compared to younger animals may be more useful in the development of a model of type-2 diabetes mellitus. Furthermore, on the basis of decrease in cholesterol concentration with age, animals fed ad libitum with possibly a high calorie diet might be even more useful in the development of a type-2 diabetes mellitus model.     

Noninvasive assessment of left ventricular and myocardial contractility in middle-aged men and women: disparate evolution above the age of 50?

End-systolic elastance (E(es)) is a frequently used index of left ventricular (LV) contractility. However, because of its inherent dependence on LV geometry, E(es) cannot be used to compare myocardial contractile state between ventricles with different geometries, which is the case in any cross-sectional study. Various normalization methods for E(es) have been proposed in the literature, but a standardized method is still lacking. In this study, we introduced a novel alternative normalization technique and compared it with three previously suggested methods. We tested all normalization methods to assess the age- and sex-related differences in myocardial contractility in a large population sample of 2,184 middle-aged (ages, 35-55 yr) untreated subjects free from overt cardiovascular disease. Ventricular contractility E(es) was determined using a previously validated noninvasive single-beat method, based on two-dimensional echocardiographic and brachial blood pressure measurements. Myocardial contractility was estimated as 1) E(es).end-diastolic volume (EDV); 2) E(es).LV mass (LVM); 3) 0.433.E(es).LVM/relative wall thickness (RWT), based on a theoretical LV model; and 4) 0.0941.E(es).LVM(0.455).RWT(-0.159), a novel semiempirical expression derived in this study. Because of the difference in their underlying assumptions, the various myocardial contractility indexes do not provide consistent information with respect to sex differences. Despite these discrepancies, it was found that myocardial contractility in women appears to be better preserved after the age of 50 yr compared with that in men. The physiological mechanisms behind this potentially clinically important phenomenon at population level require further investigation.