Variation of actin filament length in dogs.

As the smallest functional unit of force production, the sarcomeres are important in determining muscle function. Actin filament lengths, which are important in determining optimal sarcomere length for a species, have not yet been reported in dogs. This study aims to provide a species-specific value for actin filament length in dogs, while examining intraspecies, intermuscular and intramuscular variations. Muscle samples were taken from the tibialis anterior muscle, the lateral gastrocnemius muscle head and the medial gastrocnemius muscle head in 10 dogs including a Labrador, a Belgian Malinois, a Caucasian Shepherd, German Shepherds and some mixed-breed dogs. Actin filament lengths were determined through transmission electron microscopy. Mean actin filament length across all muscle samples was 0.90 ± 0.01 µm. The low intraspecies variation, combined with a lack of important inter- and intramuscular variation found in this study, affirms the use of a species-specific optimal sarcomere length of 1.89 µm for canine muscles.

Effects of overconditioning on pancreatic insulin secretory capacity, fat infiltration, and the number and size of islets in dairy cows at the end of the dry period.

In the present study, we tested the hypothesis that overconditioning in dairy cows at the end of the dry period leads to infiltration of fat and alterations of the insulin secretory capacity of the pancreas. Pregnant Holstein Friesian dairy cows were selected based on body condition score (BCS) at the start of the dry period. Body condition score varied between cows to have optimal conditioned (2.5 < BCS ≤3.5, n = 5) and overconditioned (3.5 < BCS ≤5, n = 5) cows. All animals underwent an intravenous glucose tolerance test (IVGTT) at an average of 260 d of gestation to measure the pancreatic insulin secretory capacity and assess peripheral insulin sensitivity regarding glucose metabolism. Eight days after the IVGTT, animals were slaughtered. The pancreas was dissected and weighed and tissue samples were taken for histological analysis. Results revealed that overconditioning in dairy cows led to fat infiltration in the pancreas and an increase in size of pancreatic islets expressed relative to the total area of pancreatic tissue. In addition, results revealed a positive correlation between serum fatty acid concentration and peak insulin concentration and area and number of pancreatic islets expressed relative to the total area of pancreatic tissue. The IVGTT revealed that overconditioned animals have a higher insulin secretory capacity of the pancreas, as demonstrated by higher peak insulin concentration, higher acute insulin response to glucose, and higher area under the curve (AUC) for insulin compared with optimal conditioned cows. A higher AUC for glucose during the first 60 min following administration of the glucose bolus in overconditioned cows indicates an insulin-resistant state regarding glucose metabolism. Our results suggest that the pancreas of overconditioned dairy cows at the end of gestation compensates for the concomitantly elevated level of peripheral insulin resistance by greater secretion of insulin.

Influence of adipocyte size and adipose depot on the number of adipose tissue macrophages and the expression of adipokines in dairy cows at the end of pregnancy.

The aim of this study was to determine the number of adipose tissue macrophages (ATM) and the mRNA expression of adipokines [adiponectin (ADIPOQ), leptin (LEP), interleukin 6 (IL6), tumor necrosis factor (TNF), and interleukin 10 (IL10)] in different adipose depots from cows with a variable body condition score (BCS) at the end of the dry period. We hypothesized that the number of ATM and the expression of these adipokines depend on adipocyte size and the anatomical location of the adipose depot. Subcutaneous, omental, mesenteric, perirenal, and intrapelvic adipose tissue samples were taken immediately after euthanasia of 10 Holstein Friesian dairy cows (upcoming parity 2 to 5, age 3.9 ± 1.4 yr; mean ± standard deviation) at the end of pregnancy (actual days of pregnancy at the moment of euthanasia: 269 ± 5 d). During the dry period, all animals received similar diets to meet but not exceed requirements. Five animals were considered to have a normal BCS (2.5-3.5) and 5 animals were considered to be over-conditioned (BCS = 3.75-5). Body weight of the animals at the moment of euthanasia was 717 ± 77 kg. Expression of the different genes was determined by reverse transcription quantitative real-time PCR. Adipocyte size was determined by measuring the area of 100 adipocytes on histological sections. Average adipocyte area was 10,475 ± 1,019, 8,500 ± 780, 10,383 ± 1,227, 11,466 ± 1,039, and 11,087 ± 1,632 µm2 for the subcutaneous, mesenteric, omental, intrapelvic, and perirenal adipose depot, respectively. Immunohistochemistry using anti-bovine CD172a antibodies was performed to determine the proportion of ATM (the number of CD172a-positive cells per 100 adipocytes, given as a percentage). Expression of LEP, IL6, and TNF was positively associated with adipocyte size, whereas no association could be detected between ADIPOQ and IL10 with the size of the adipocytes. The omental adipose depot was especially infiltrated with ATM (1.92 ± 0.55, 1.10 ± 0.33, and 8.28 ± 2.24% for the subcutaneous, mesenteric, and omental adipose depot, respectively). The proportion of ATM was positively associated with the size of the adipocytes in the omental and mesenteric adipose depot. Expression of ADIPOQ, LEP, IL6, TNF, and IL10 differed among depots, which suggests differences in inflammatory characteristics depending on the anatomical location of depots. In conclusion, the results of the present study confirm the adipose tissue as a potential source of inflammatory mediators and demonstrate ATM infiltration, especially in the omental adipose depot.

Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer-assisted image analysis.

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer-assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm® software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B-cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F-cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA:TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre-emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.

Use of computer-assisted image analysis for semi-quantitative histology of the hepatopancreas in whiteleg shrimp Penaeus vannamei (Boone).

Despite the increasing use of novel molecular techniques in pathology, histology remains the standard method for monitoring tissue alterations and for assessing pathology. Histopathological evaluation is generally laborious and subjective with risk of discrepancies in semi-quantitative scoring between pathologists. In contrast, computer-assisted image analysis (CAIA) is potentially faster, more objective and thus suitable for routine screening. Limited research has been carried out on CAIA in crustacean histopathology, and the methods described were not fully automated. Therefore, the aim of this study was to develop CAIA in whiteleg shrimp (Penaeus vannamei) for the study of the hepatopancreas. Paraffin sections were immunohistochemically stained with monoclonal antibodies WSH8 against haemocytes and counterstained with Mayer's haematoxylin for detection of haemocytes and B-cell vacuoles, and modified toluidine blue protocol was used for detection of F-cells; frozen sections were stained with Oil Red O for detection of lipid droplets within R-cells. Visiopharm® software was used to develop and validated protocols for the quantification of morphological parameters (areas of haemocyte infiltration, F-cells, B-cell vacuoles, lipid droplets and their ratios to total tissue area and total lumen area). These protocols enable the future use of CAIA for determination of the nutritional and pathological condition of this organ.

Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp, Penaeus vannamei (Boone).

Pathology in penaeid shrimps relies on histology, which is subjective, time-consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.

Short communication: Limitations of glucose tolerance tests in the assessment of peripheral tissue insulin sensitivity during pregnancy and lactation in dairy heifers.

The aim of the present study was to point at the limitations of glucose tolerance tests (GTT) to assess peripheral tissue insulin sensitivity in dairy heifers in different physiological states (pregnancy and lactation). Intravenous GTT were performed in 5 nonpregnant, nonlactating heifers, 5 heifers at the end of pregnancy (12-7 d before calving), and 5 lactating primiparous cows (11-14 d after calving). Glucose and insulin concentrations were determined and area under the curve (AUC) and clearance rate of glucose and insulin were calculated. Additionally, data were analyzed using the minimal model to derive the insulin sensitivity parameter (Si). Basal glucose and insulin concentrations were greater in the nonpregnant, nonlactating heifers. Clearance rate of glucose and Si were lowest, whereas the AUC for glucose was greatest in the nonpregnant, nonlactating heifers. Insulin concentrations during the GTT were greater for the nonpregnant, nonlactating heifers. Results from the GTT in pregnant heifers and lactating primiparous cows are biased by the fact that a large part of the glucose disappearance during an intravenous GTT occurs independently of insulin by the pregnant uterus or the lactating mammary gland. As such, greater AUC of glucose, lower clearance rate of glucose, or lower Si derived from GTT performed in nonpregnant, nonlactating dairy heifers in the present study might indicate decreased peripheral tissue insulin sensitivity of the glucose metabolism or decreased insulin-independent glucose disappearance. Based on the results from a GTT, it is impossible to discriminate between both metabolic pathways. It can be concluded that parameters derived from GTT are not suited to compare peripheral tissue insulin sensitivity of the glucose metabolism between dairy heifers in different physiological states due to the large variation in insulin secretion and the substantial difference in insulin-independent glucose disposal associated with these physiological states.

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions.

A thorough understanding of host-microbe interactions is crucial for more efficient disease management in the marine larviculture industry. As demonstrated in terrestrial animal research, gnotobiotic systems (involving animals cultured in germ-free conditions or inoculated with known microorganisms) are excellent tools to extend our understanding of the mechanisms involved in host-microbe interactions and allow the evaluation of new treatments for diseases. In this study, we introduce a germ-free European sea bass Dicentrarchus labrax larval model, independent of the continuous addition of antimicrobial agents. This model has an experimental set-up that allows addition of live feed to the larvae without compromising the germ-free status. This model will facilitate and render aquaculture research more effective in terms of mitigation fish larval diseases.

Influence of adipocyte size and adipose depot on the in vitro lipolytic activity and insulin sensitivity of adipose tissue in dairy cows at the end of the dry period.

The aim of the present research was to describe characteristics of adipose tissue lipolysis in dairy cows with a variable body condition score (BCS). Ten clinically healthy Holstein Friesian cows were selected based on BCS and euthanized 10 to 13 d before the expected parturition date. Immediately after euthanasia, adipose tissue samples were collected from subcutaneous and omental fat depots. In both depots, we observed an increase in adipocyte size with increasing BCS. Using an in vitro explant culture of subcutaneous and omental adipose tissue, we aimed to determine the influence of adipocyte size and localization of adipose depot on the lipolytic activity in basal conditions and after addition of isoproterenol (nonselective ß-agonist) and insulin in different concentrations. Glycerol release in the medium was used as a measure for lipolytic activity. We observed that the basal lipolytic activity of subcutaneous and omental adipose tissue increased with adipocyte volume, meaning that larger fat cells have higher basal lipolytic activity independent of the location of the adipose depot. Dose-response curves were created between the concentration of isoproterenol or insulin and the amount of glycerol released. The shape of the dose-response curves is determined by the concentration of isoproterenol and insulin needed to elicit the half-maximal effect and the maximal amount of stimulated glycerol release or the maximal inhibitory effect of insulin. We observed that larger fat cells released more glycerol upon maximal stimulation with isoproterenol and this was more pronounced in subcutaneous adipose tissue. Additionally, larger fat cells had a higher sensitivity toward lipolytic signals. We observed a trend for larger adipocytes to be more resistant to the maximal antilipolytic effect of insulin. The insulin concentration needed to elicit the half-maximal inhibitory effect of insulin was within the physiological range of insulin and was not influenced by adipocyte size or adipose depot. We conclude that overconditioned cows have larger adipocytes and are predisposed to excessive mobilization of body fat due to a higher basal and stimulated lipolytic activity of large adipocytes while the antilipolytic effect of insulin is preserved.

Insulin response of the glucose and fatty acid metabolism in dry dairy cows across a range of body condition scores.

The objective of the present research was to determine the insulin response of the glucose and fatty acid metabolism in dry dairy cows with a variable body condition score (BCS). Ten pregnant Holstein Friesian dairy cows (upcoming parity 2 to 5) were selected based on BCS at the beginning of the study (2mo before expected parturition date). During the study, animals were monitored weekly for BCS and backfat thickness and in the last 2wk, blood samples were taken for determination of serum nonesterified fatty acid (NEFA) concentration. Animals underwent a hyperinsulinemic euglycemic clamp test in the third week before the expected parturition date. The hyperinsulinemic euglycemic clamp test consisted of 4 consecutive insulin infusions with increasing insulin doses: 0.1, 0.5, 2, and 5mIU/kg per minute. For each insulin infusion period, a steady state was defined as a period of 30min where no or minor changes of the glucose infusion were necessary to keep the blood glucose concentration constant and near basal levels. During the steady state, the glucose infusion rate [steady state glucose infusion rate (SSGIR) in µmol/kg per minute] and NEFA concentration [steady state NEFA concentration (SSNEFA) in mmol/L] were determined and reflect the insulin response of the glucose and fatty acid metabolism. Dose response curves were created based on the insulin concentrations during the steady state and the SSGIR or SSNEFA. The shape of the dose response curves is determined by the concentration of insulin needed to elicit the half maximal effect (EC50) and the maximal SSGIR or the minimal SSNEFA for the glucose or fatty acid metabolism, respectively. The maximal SSGIR was negatively associated with variables reflecting adiposity of the cows (BCS, backfat thickness, NEFA concentration during the dry period, and absolute weight of the different adipose depots determined after euthanasia and dissection of the different depots), whereas the EC50 of the glucose metabolism was positively associated with these variables. These results reflect a decreased insulin sensitivity and a decreased insulin responsiveness of the glucose metabolism in overconditioned dry dairy cows. The minimal SSNEFA and the EC50 of the fatty acid metabolism were not associated with variables reflecting adiposity of the cows, meaning that the insulin response of the fatty acid metabolism was not associated with the level of fat accumulation in dry dairy cows. Additionally, within individual cows, the EC50 of the glucose metabolism was higher than the EC50 of the fatty acid metabolism, meaning that the response of the fatty acid metabolism occurs at lower insulin concentrations compared with the response of the glucose metabolism. It can be concluded that a negative association exists between the level of fat accumulation in pregnant dairy cows at the end of the dry period and the insulin response of the glucose metabolism.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi.

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1- and Th2-type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial-derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ-T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA-3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

A field study to unravel factors that are significantly associated with the secretory activity of the corpus luteum during the first three postpartum cycles in high yielding dairy cows, based on the amount of steroidogenic and endothelial cells present in the luteal tissue.

Fourteen multi- and eight primiparous high-yielding dairy cows were followed from the first till the fourth ovulation postpartum. Cows were randomly divided into two groups and supplemented with soybean (group I; n = 11) or rapeseed meal (group II; n = 11). Both groups were subjected to a biopsy sampling of the corpus luteum (CL) at cycle day 9. The luteal capillary network (visualized by Bandeiraea simplicifolia) was denser in cycles 2 and 3 (p = 0.0005). The same was seen for the surface occupied by steroidogenic cells (visualized by 3ß-hydroxysteroiddehydrogenase) (p = 0.0001). The peripheral blood progesterone concentration showed an increasing trend with increasing cycle number and was higher in primiparous cows (p = 0.013), which had also larger glands on cycle day 9. The area occupied by endothelial cells was positively correlated with the area occupied by steroidogenic cells (r = 0.59; p < 0.0001). Both the areas occupied by endothelial and by steroidogenic cells were negatively correlated with the blood concentration of nonesterified fatty acids (NEFAs) (respectively, r = -0.377; p = 0.004 and r = -0.355; p = 0.007). We can conclude that primiparous cows generally have higher peripheral progesterone levels during the first three cycles after calving which is associated with a larger CL. In comparison with those of the first post-partum cycle, corpora lutea of cycles 2 and 3 have a denser capillary network and a larger area of steroidogenic cells, while these are only associated with a trend of higher peripheral progesterone concentrations.

Development of a three-dimensional computer model of the equine heart using a polyurethane casting technique and in vivo contrast-enhanced computed tomography.

INTRODUCTION/OBJECTIVES: Insight into the three-dimensional (3D) anatomy of the equine heart is essential in veterinary education and to develop minimally invasive intracardiac procedures. The aim was to create a 3D computer model simulating the in vivo anatomy of the adult equine heart. ANIMALS: Ten horses and five ponies. MATERIALS AND METHODS: Ten horses, euthanized for non-cardiovascular reasons, were used for in situ cardiac casting with polyurethane foam and subsequent computed tomography (CT) of the excised heart. In five anaesthetized ponies, a contrast-enhanced electrocardiogram-gated CT protocol was optimized to image the entire heart. Dedicated image processing software was used to create 3D models of all CT scans derived from both methods. Resulting models were compared regarding relative proportions, detail and ease of segmentation. RESULTS: The casting protocol produced high detail, but compliant structures such as the pulmonary trunk were disproportionally expanded by the foam. Optimization of the contrast-enhanced CT protocol, especially adding a delayed phase for visualization of the cardiac veins, resulted in sufficiently detailed CT images to create an anatomically correct 3D model of the pony heart. Rescaling was needed to obtain a horse-sized model. CONCLUSIONS: Three-dimensional computer models based on contrast-enhanced CT images appeared superior to those based on casted hearts to represent the in vivo situation and are preferred to obtain an anatomically correct heart model useful for education, client communication and research purposes. Scaling was, however, necessary to obtain an approximation of an adult horse heart as cardiac CT imaging is restricted by thoracic size.

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species.

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards - amongst others - ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

The bovine luteal histological composition: a topographic point of view.

High-yielding dairy cows are struggling with a high incidence of embryonic loss, among others caused by an insufficient peripheral progesterone concentration which for its part might be associated with an impaired luteal progesterone production. This impaired capacity to produce progesterone might be reflected in the histology of the gland. The aim of the present pilot study was the assessment of the variation in cell density within a bovine luteal gland (LG), to examine whether it is possible to analyse histologically the functionality of the gland based on one single tissue sample. Six LGs (stage II or III) were harvested out of just as many healthy cows at the slaughterhouse. The luteal cell density was assessed by calculating the nuclear density (ND) of the different luteal cell types on haematoxylin-eosin-stained histological sections from a number of topographic regions evenly spread throughout the glands, to give an overview of the pattern of cellular distribution within the whole gland. Cells were differentiated into 'large luteal cells', 'small luteal cells' and 'non-steroidogenic cells'. Results show that the cellular density, within a tissue sample is not significantly influenced by its location in relation to the gland's equatorial plane. However, the position with respect to the polar axis of the gland has a decisive effect, as the ND is significantly higher (p < 0.05) in the peripheral regions (outer zone) when compared with the central regions (inner zone) of the gland, and this counts for all three cell types.

Topographic distribution of the different cell types, connective tissue and vascular tissue/lumina within a functional bovine corpus luteum and its association with breed, type of fixation protocol and stage during the cycle.

In the present study, we analysed the effect of fixative, breed, luteal stage and location on the nuclear density, volume density of connective tissue and vascular tissue/lumina within a bovine luteal gland in view of the development of an in vivo sampling technique to longitudinally monitor luteal histophysiology. The inner zone defined as the zone geometrically closest to the centre of the gland shows a significantly lower nuclear density (for all cell types) and a higher volume density of collagen fibres and vessels when compared with the outer zone (p < 0.001). The nuclear density in luteal glands from Holstein-Friesian cows is not significantly different from that in Belgian Blue cows, nor is it in stage II vs stage III glands. The collagen fibre content was significantly lower in glands of Belgian Blue cows (p = 0.01) and in younger glands (p = 0.003). Hence, it seems that the lower nuclear density in the inner zone was compensated by a higher amount of collagen fibres. As the type of fixative applied has a significant effect on the nuclear density of the different cell types, the present study warrants future research to further optimize the fixation protocol. As a conclusion, we can state that the topographic difference in nuclear distribution for the different cell types in a bovine luteal gland is only significant when comparing the inner vs the outer zone. This implies that if a sample representative for the whole gland has to be taken, for example, when taking an in vivo sample, it is necessary that the biopsy goes through the inner zone and contains the total diameter of the gland.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae.

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Stereology and computer assisted three-dimensional reconstruction as tools to study probiotic effects of Aeromonas hydrophila on the digestive tract of germ-free Artemia franciscana nauplii.

AIMS: Validation of stereology and three-dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ-free Artemia franciscana nauplii. METHODS AND RESULTS: Germ-free Artemia nauplii were cultured using Baker's yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three-dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three-dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely. CONCLUSION: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three-dimensional reconstruction. SIGNIFICANCE AND IMPACT OF THE STUDY: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro-organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three-dimensional reconstruction are prerequisite to obtain more powerful analysis.

Feasibility of the Ussing chamber technique for the determination of in vitro jejunal permeability of passively absorbed compounds in different animal species.

The aim of this study was to assess the feasibility of the Ussing chamber technique for the determination of the jejunal permeability of passively absorbed, high permeability model compounds (acetaminophen and ketoprofen) in different animal species. Additionally, electrophysiological measurements and histological examination of pre- and post-incubation tissue specimens were performed. Apparent permeability coefficients of turkey and dog jejunum were low and highly variable due to tissue fragility caused by differences in thickness of the remaining intestinal layers after stripping and resulting in severe damage. Pig and horse jejunum were markedly more suitable for permeability determinations and mild signs of deterioration were noticed after 120 min of incubation. Transepithelial electrical resistance and potential difference did not correlate well with the observed tissue damage. From these data, the Ussing chamber technique appears to allow for permeability measurements within a species, but seems unsuitable for interspecies permeability comparison. However, further validation of the method with low permeability compounds and actively transported compounds is needed.