Mimicking the temperature gradient between the sow's oviduct and uterus improves in vitro embryo culture output.

This work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on ARTs. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturized probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in vivo values, IVF was performed at three temperature conditions (37.0°C, 38.5°C and 39.5°C) and presumptive zygotes were cultured in these conditions for 20 h, while further embryo culture (EC) (21-168 h post-insemination) was maintained at 38.5°C. After 20 h, different fertility parameters were assessed. During EC, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5°C was found between the oviduct and uterus of sows (P < 0.05) and when this gradient was transferred to pig in vitro culture, the number of poly-nuclear zygotes after IVF was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0°C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0°C compared with those of sperm incubated at higher temperatures (38.5°C and 39.5°C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.

Plastinated macroparasites, an alternative resource for use in practical lessons.

The plastination technique is a recently developed option to preserve biological tissues widely used in human and veterinary anatomy. This process allows one to obtain real, safe, clean, dry, resistant and permanently stable material without any additional treatment or maintenance. All these advantages are associated with the rapid expansion of plastination during recent decades in a high number of educational institutions. However, the studies of parasite conservation through plastination are scarce, although the material obtained has a high quality, as prior references have shown. In this study, 141 students of veterinary medicine and pharmacy university degrees used, in a blind study, plastinated specimens (experimental group) or wet specimens (control group) during three consecutive practical sessions. Afterwards all the students completed anonymous questionnaires on both knowledge and satisfaction. The results demonstrated that plastinated parasites are a valid and satisfactory alternative to be used as a teaching/learning tool during practical lessons. Moreover, with regard to the knowledge of morphological structures, the results from the questionnaire of assimilated knowledge showed no differences between the use of plastinated and formaldehyde-preserved parasites.

La technique de plastination est une méthode assez récente de préservation des tissus biologiques qui est désormais largement utilisée en anatomie humaine et vétérinaire. Ce procédé permet d'obtenir des préparations anatomiques réalistes, sûres, sèches, résistantes et stables qui ne nécessiteront pas de traitement ni d'entretien ultérieurs pour préserver leur durabilité. Ces avantages expliquent que le recours à la plastination se soit rapidement développé au cours des dernières décennies dans nombre d'institutions d'enseignement. Néanmoins, rares sont les études portant sur la préservation des parasites par plastination, bien que les spécimens obtenus lors de travaux antérieurs aient été jugés de très bonne qualité. Les auteurs présentent les résultats d'une enquête menée auprès de 141 étudiants préparant leur diplôme de médecine vétérinaire ou de pharmacie, à qui ont été distribués en aveugle des spécimens plastinés (groupe expérimental) et des spécimens conservés dans une solution liquide (groupe de contrôle) lors de trois séances consécutives de travaux pratiques. Il a ensuite été demandé aux étudiants de remplir anonymement un questionnaire portant sur les connaissances acquises lors de ces séances et sur leur degré de satisfaction. Les résultats ont montré que les parasites plastinés constituent une alternative valable et satisfaisante en tant que matériel pédagogique et d'entraînement pendant les leçons pratiques. Par ailleurs, s'agissant de l'étude des structures morphologiques, les résultats du questionnaire sur les connaissances acquises lors des travaux pratiques n'ont pas fait apparaître de différences entre les étudiants ayant travaillé sur des parasites plastinés et ceux ayant travaillé avec des parasites conservés dans le formol.

La técnica de la plastinación es un método de reciente aparición para conservar tejidos biológicos, utilizado con gran frecuencia en anatomía humana y veterinaria. Se trata de un proceso que permite obtener material real, inocuo, limpio, seco, resistente y con una estabilidad permanente sin necesidad de ninguna otra operación de tratamiento o mantenimiento. Todas estas ventajas explican que en los últimos decenios la técnica se haya extendido con rapidez por muy numerosas instituciones de enseñanza. Aun así, hay escasos estudios sobre la conservación de parásitos por plastinación, pese a que el material obtenido, como se desprende de referencias anteriores, es de gran calidad. Los autores describen un estudio ciego en el que 141 estudiantes universitarios en farmacia y medicina veterinaria emplearon muestras plastinadas (grupo experimental) o muestras húmedas (grupo de control) en tres clases prácticas consecutivas, tras lo cual todos los estudiantes respondieron anónimamente a un cuestionario que evaluaba tanto sus conocimientos como su nivel de satisfacción. Los resultados demostraron que los parásitos plastinados constituyen una alternativa válida y satisfactoria como herramienta para impartir y/o recibir clases prácticas. Además, desde el punto de vista del conocimiento de estructuras morfológicas, los resultados del cuestionario sobre conocimientos asimilados no pusieron de manifiesto diferencia alguna entre el uso de parásitos plastinados y el de parásitos conservados en formaldehído .

Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig.

STUDY QUESTION: Is O2 tension in the pig oviduct and uterus affected by the estrous cycle stage and the animal's age, and can the outcome of in vitro embryo development be improved by mimicking these physiological values? SUMMARY ANSWER: O2 tension within the pig reproductive organs is affected by the animal's age, and values close to those measured in vivo have a positive impact on embryo development and quality when used during IVF and embryo culture (EC). WHAT IS KNOWN ALREADY: To obtain a healthy embryo in vitro, it is necessary to adopt a culture microenvironment that approximates physiological conditions. Despite advances in surgical procedures and sensitive probes that allow accurate assessment of in vivo O2 tension, few such studies have been conducted recently in mammals. In addition, no reference values of physiological O2 tension in the reproductive tract exist for large animal models such as pig, and the effect of O2 tension on ART outcomes is unknown. STUDY DESIGN, SIZE, DURATION: This study was conducted in pigs. We measured oviductal and uterine O2 tension (n = 29 and 13, respectively) and then examined how the use of the physiological values in pig IVF and EC affected pig ART output (n = 1447 oocytes). PARTICIPANTS/MATERIALS, SETTING, METHODS: The oviductal and uterine O2 tension at the different stages of the estrous cycle was monitored using a laparo-endoscopic single-site surgery (LESS) assisted approach along with a flexible and thin miniaturized luminescent probe. Two groups of pigs, Large-white × Landrace breed, were used: for the first group, 16 pre-pubertal gilts (5 months old and 95 kg) were induced to ovulate with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); in the second group 13 mature sows (24-48 months and 185 kg) were used. IVF and EC were performed at two different O2 tensions: Atmospheric O2 (20%) and the mean in vivo value measured (7%). At 18-20 h post-insemination (hpi), a small sample of presumptive zygotes were fixed, stained and examined under epifluorescence microscopy to assess the fertilization rates. At 48 hpi, cleavage was evaluated under the stereomicroscope. Finally, at 180 hpi, development to the blastocyst stage was quantified, blastocyst morphology was assessed, and embryos were fixed and stained to count the mean cell number per blastocyst. MAIN RESULTS AND THE ROLE OF CHANCE: The mean O2 content within the pig oviduct and uterus was always lower than in ambient air. The average O2 percentage was higher in gilts (10.0%) than in sows (7.6%) (P < 0.0001). The cleavage rate of porcine in vitro fertilized embryos maintained under 7% O2 during IVF and EC was significantly higher (60.0 ± 2.3) compared with those cultured under 20% O2 (32.0 ± 2.2) (P < 0.05). An increase in the number of cells in embryos cultured under the low O2 concentration (88.9 ± 5.9) was observed compared to those cultured under 20% O2 (59.0 ± 5.0) (P < 0.05). LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although minimally invasive surgery was used the effect of anesthesia and manipulations on O2 tension within the organs are unknown. WIDER IMPLICATIONS OF THE FINDINGS: Using physiological oxygen concentrations in IVF/EC could improve ART outcomes. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Grants AGL2012-40180-C03-01 and AGL2015-66341-R. The authors declare no conflict of interest.

Temperature gradients in vivo influence maturing male and female gametes in mammals: evidence from the cow.

Since 1980 several reports have indicated that temperatures vary between preovulatory follicles and other ovarian tissues in rabbit, cow, pig and human. However, these observations did not achieve prominence; they were regarded as artefacts due to the use of anaesthetics and open surgery (laparotomy). Recently, without resorting to anaesthesia or surgery, direct measurements of temperature in preovulatory follicles have been performed in the cow by means of a thermistor probe introduced into the antrum under ultrasonic guidance. Such follicles revealed a mean antral (follicular fluid) temperature 0.74°C and 1.54°C cooler than uterine surface and rectal temperatures respectively in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. Cows are predominantly monovular and preovulatory follicles attain a diameter of 15-22mm or more. These features and the timescale of response to the preovulatory gonadotrophin surge make them a valuable model for the human preovulatory follicle. Temperature gradients are interpreted primarily in a context of final maturation of gametes immediately before the onset of fertilisation. Preovulatory follicular temperature in women could be assessed by a comparable approach and might become a valuable selection guide for oocyte viability.

Effects of pneumoperitoneum and body position on the morphology of the caudal cava vein analyzed by MRI and plastinated sections.

BACKGROUND: Pneumoperitoneum and patient positioning are essential factors during laparoscopic surgical procedures. They cause hemodynamic and anatomical changes in several abdominal organs among which the caudal cava vein (CCV) is involved. Hemodynamic changes in this vein (decreased venous return) have been described in the porcine model, but how the vein morphology and size is affected at different abdominal levels is unknown. We sought to assess the morphological and morphometrical changes in the CCV of the pig caused by pneumoperitoneum and the reverse Trendelenburg position by in vivo magnetic resonance imaging (MRI). METHODS: Six pigs were scanned via MRI under four situations: S1, control (no pneumoperitoneum); S2, control in the reverse Trendelenburg position; S3, pneumoperitoneum (14 mmHg); and S4, pneumoperitoneum in the reverse Trendelenburg position. MRI and plastinated body sections were used to evaluate the topography, morphology and cross-sectional area of the CCV. RESULTS: Two portions of the CCV were differentiated: a prehepatic portion (located between the vertebral levels L1-T15) with flat and irregular morphology, and a hepatic portion (between T14-T11) that was almost rounded. The reverse Trendelenburg position caused an increase in the lumen affecting mainly the prehepatic portion, while pneumoperitoneum caused a decrease in the total vascular lumen, exerting a greater effect on the hepatic portion. The combination of both situations resulted in a further decrease in the vascular area and global morphological changes. CONCLUSIONS: The pneumoperitoneum and reverse Trendelenburg position caused morphological and morphometrical changes in the prehepatic and hepatic portions of the CCV, which should assist in gaining a better understanding of the hemodynamic changes described in the literature.

Validity of insertion depth measurement in double-balloon endoscopy.

BACKGROUND AND STUDY AIMS: In double-balloon enteroscopy (DBE) the use of a reliable and practical method to calculate the insertion depth of the endoscope could help to improve diagnosis accuracy and optimize the effort and cost of the technique. The objectives of this work were to compare and evaluate two methods of estimating the insertion depth and to obtain a descriptive model capable of representing the exploration dynamics and efficiency in terms of advanced distance and time. METHODS: Oral DBE was performed in 25 pigs. Insertion depth was calculated during the procedure by: 1) estimation of time and distance for each push and pull cycle during progression; and 2) estimation of distance during withdrawal. At the maximum insertion depth a tattoo was placed, and the observed measures for the two methods were compared with the distance between the pylorus and the mark after euthanasia and necropsy of the animals 1 week after DBE. RESULTS: The average insertion depth during progression, withdrawal, and after necropsy was 324.92 cm, 317.23 cm, and 342.05 cm, respectively (P Anova = 0.72). The Pearson correlation coefficient (r > 0.85; P < 0.001) and paired Brand - Altman plots demonstrated high agreement between progression and necropsy (0.03 % difference) and between withdrawal and necropsy (6.9 % difference). The exploration dynamics and efficiency in terms of advanced distance per cycle and time fitted to potential and logarithmic regression models, respectively. CONCLUSIONS: Measurement of insertion depth in vivo was validated in the porcine model during progression and withdrawal. Estimation during progression was more accurate and allowed exploration dynamics and efficiency to be plotted, which might be used as approximate reference values for humans.

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

BACKGROUND: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. RESULTS: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. CONCLUSIONS: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

Magnetic resonance imaging of the equine temporomandibular joint anatomy.

REASONS FOR PERFORMING STUDY: In human medicine, magnetic resonance imaging (MRI) is considered the 'gold standard' imaging procedure to assess the temporomandibular joint (TMJ). However, there is no information regarding MRI evaluation of equine TMJ. OBJECTIVES: To describe the normal sectional MRI anatomy of equine TMJ by using frozen and plastinated anatomical sections as reference; and determine the best imaging planes and sequences to visualise TMJ components. METHODS: TMJs from 6 Spanish Purebred horse cadavers (4 immature and 2 mature) underwent MRI examination. Spin-echo T1-weighting (SE T1W), T2*W, fat-suppressed (FS) proton density-weighting (PDW) and fast spin-echo T2-weighting (FSE T2W) sequences were obtained in oblique sagittal, transverse and dorsal planes. Anatomical sections were procured on the same planes for a thorough interpretation. RESULTS: The oblique sagittal and transverse planes were the most informative anatomical planes. SE T1W images showed excellent spatial resolution and resulted in superior anatomic detail when comparing to other sequences. FSE T2W sequence provided an acceptable anatomical depiction but T2*W and fat-suppressed PDW demonstrated higher contrast in visualisation of the disc, synovial fluid, synovial pouches and articular cartilage. CONCLUSIONS: The SE T1W sequence in oblique sagittal and transverse plane should be the baseline to identify anatomy. The T2*W and fat-suppressed PDW sequences enhance the study of the articular cartilage and synovial pouches better than FSE T2W. POTENTIAL RELEVANCE: The information provided in this paper should aid clinicians in the interpretation of MRI images of equine TMJ and assist in the early diagnosis of those problems that could not be diagnosed by other means.

Morphological changes in the porcine cervix: A comparison between nulliparous and multiparous sows with regard to post-cervical artificial insemination.

In recent decades, new artificial insemination (AI) methods, such as post-cervical AI (PCAI), have been developed in pig. PCAI involves crossing the cervix to deposit the sperm in the uterine body. Although PCAI application in sows is frequent, its application in nulliparous (gilts) females it is still limited due to the difficulty of passing through the cranial part of the cervical lumen. We hypothesized that ageing and parity would modify the cervical canal, facilitating the introduction of AI devices through the cervix. The aim was to compare the morphology of the uterus at different levels between multiparous and nulliparous females. Morphological analysis of the uterus pointed to a longer cervix (25.9 ±â€¯4.6 vs. 21.6 ±â€¯3.3 cm, p < 0.001) and greater length of the part of the reproductive tract involved in PCAI (from rima vulvae to the last cervical cushion) (56.2 ±â€¯6.0 vs. 50.3 ±â€¯5.2 cm, p < 0.001) in multiparous sows compared with nulliparous animals. As regards the structure of the vaginal and uterine parts of the cervix (the part in contact with the vagina and uterine body, respectively), the cross-sectional area, perimeter and total thickness were greater in the uterine part of multiparous than of nulliparous animals (area: 4.07 ±â€¯1.46 vs. 2.46 ±â€¯0.56 cm2, p < 0.01; perimeter: 8.50 ±â€¯1.44 cm vs. 6.28 ±â€¯0.92 cm, p < 0.001; thickness: 10.79 ±â€¯0.96 vs. 8.35 ±â€¯0.62 mm, p < 0.05), but not in the vaginal part. The tissue content analysed in histological cross-sections also showed differences between female groups, a greater content of connective tissue (58.86 ±â€¯10.78 vs. 67.60 ±â€¯13.38%, p < 0.001) and a lower amount of muscle fibres (39.79 ±â€¯10.24 vs. 30.66 ±â€¯13.69%, p < 0.001) being observed in multiparous sows. Finally, silicone casts of the cervical lumen revealed differences between the two groups in the size and shape of the ridges in the lumen trajectory. Parity, which is also influenced by ageing, determines important changes in the size, structure and tissue content of the cervix wall, as well as in the morphology of the cervical canal, which may be responsible for the different levels of performance of PCAI in the female populations. Therefore, the future design of AI strategies and catheters should take into consideration the morphological variations of the cervix lumen, which will depend on age and parity of the females.

Computed tomographic anatomy of the temporomandibular joint in the young horse.

REASONS FOR PERFORMING STUDY: The equine temporomandibular joint (TMJ) and its surrounding structures can be difficult to investigate in cases with a clinical problem related to the region. Little previous attention has been given either to a computed tomographic (CT) imaging protocol for the joint or an interpretation of the structures displayed in CT images of the normal joint. OBJECTIVES: To provide a CT atlas of the normal cross-sectional anatomy of the equine TMJ using frozen and plastinated sections as anatomical reference. METHODS: Eight TMJs from 4 immature pure-bred Spanish horses were examined by helical CT. Scans were processed with a detailed algorithm to enhance bony and soft tissue. Transverse CT images were reformatted into sagittal and dorsal planes. Transverse, sagittal and dorsal cryosections were then obtained, photographed and plastinated. Relevant anatomic structures were identified in the CT images and corresponding anatomical sections. RESULTS: In the CT images, a bone window provided excellent bone detail, however, the soft tissue components of the TMJ were not as well visualised using a soft tissue window. The articular cartilage was observed as a hyperattenuating stripe over the low attenuated subchondral bone and good delineation was obtained between cortex and medulla. The tympanic and petrous part of the temporal bone (middle and inner ear) and the temporohyoid joint were seen in close proximity to the TMJ. CONCLUSIONS: Helical CT provided excellent images of the TMJ bone components to characterise the CT anatomy of the normal joint. POTENTIAL RELEVANCE: Detailed information is provided that may be used as a reference by equine veterinarians for the CT investigation of the equine TMJ and serve to assist them in the diagnosis of disorders of the TMJ and related structures (middle and inner ear). The study was performed at an immature stage and further studies of mature individuals are required in order to confirm that the clinical interpretation is not affected by changes occurring with age.

Expression of the vascular endothelial growth factor system (VEGF) in the porcine oviduct during the estrous cycle.

The expression and localization of VEGFA and its main receptors Flt-1 and KDR is characterized in the oviduct throughout the porcine estrous cycle. Oviducts were sampled from sows at early follicular, late follicular, early luteal and late luteal stages of the estrous cycle and a segment from the mid portion of the ampulla and isthmus studied by real time RT-PCR and quantitative immunohistochemistry. The expression of the three components of the VEGF system was continuous, although differences were observed depending on the oviduct portion, the stage of the estrous cycle and the histological layer. VEGFA and KDR mRNA expressions were higher in ampulla, while Flt-1 mRNA was higher in isthmus. VEGFA protein was also higher in ampulla but Flt-1 and KDR did not show regional differences between ampulla and isthmus. While the mRNA expression of VEGFA, Flt-1 and KDR increased progressively during the luteal period, the amount of VEGFA and Flt-1 protein decreased in the same period (in isthmus only). Contrastinly, KDR protein peaked in ampulla and isthmus just before ovulation. The VEGF system was majorly located in both the secretory and ciliated cells of the oviduct epithelium, but also in the endothelium and fibroblasts of the lamina propia and the muscle fibres and vessels of the tunica muscularis. Our results are consistent with a participation of VEFG in the regulation of the dynamics of oviductal fluid secretion and the oviduct contractibility.

Mechanical Intestinal Obstruction in a Porcine Model: Effects of Intra-Abdominal Hypertension. A Preliminary Study.

INTRODUCTION: Mechanical intestinal obstruction is a disorder associated with intra-abdominal hypertension and abdominal compartment syndrome. As the large intestine intraluminal and intra-abdominal pressures are increased, so the patient's risk for intestinal ischaemia. Previous studies have focused on hypoperfusion and bacterial translocation without considering the concomitant effect of intra-abdominal hypertension. The objective of this study was to design and evaluate a mechanical intestinal obstruction model in pigs similar to the human pathophysiology. MATERIALS AND METHODS: Fifteen pigs were divided into three groups: a control group (n = 5) and two groups of 5 pigs with intra-abdominal hypertension induced by mechanical intestinal obstruction. The intra-abdominal pressures of 20 mmHg were maintained for 2 and 5 hours respectively. Hemodynamic, respiratory and gastric intramucosal pH values, as well as blood tests were recorded every 30 min. RESULTS: Significant differences between the control and mechanical intestinal obstruction groups were noted. The mean arterial pressure, cardiac index, dynamic pulmonary compliance and abdominal perfusion pressure decreased. The systemic vascular resistance index, central venous pressure, pulse pressure variation, airway resistance and lactate increased within 2 hours from starting intra-abdominal hypertension (p<0.05). In addition, we observed increased values for the peak and plateau airway pressures, and low values of gastric intramucosal pH in the mechanical intestinal obstruction groups that were significant after 3 hours. CONCLUSION: The mechanical intestinal obstruction model appears to adequately simulate the pathophysiology of intestinal obstruction that occurs in humans. Monitoring abdominal perfusion pressure, dynamic pulmonary compliance, gastric intramucosal pH and lactate values may provide insight in predicting the effects on endorgan function in patients with mechanical intestinal obstruction.

The histochemical profiles of fibre types in porcine skeletal muscle.

Using a variety of histochemical methods -mATPase staining after alkaline and acid preincubations, NADH-TR and alpha-MGPDH- we have investigated the fibre types in porcine skeletal muscle. The results reveal that four major fibre types -I, IIA, IIB and II*- can be separated histochemically in Longissimus lumborum muscle of Landrace pigs. The histochemical properties of the muscle fibre type II* are very similar to that of type IIX described in other mammals. The existence of IIX fibres in pig muscle has been recently demonstrated by molecular biology techniques and our results validate the use of histochemistry (mATPase) as an easy methodology to differentiate the three fast myosins (type II fibres) in pig muscle.

Postnatal development of semitendinosus muscle in the dog.

In this study the differentiation of postnatal muscle fibres in dog semitendinosus muscle has been characterized. Several histochemical techniques for myosin ATPase and metabolic activity were used in animals aged between 1 day and 2 months. The results show that at birth there are two types of fibre, whose ATPase activity gradually changes during postnatal development, so that several types of fibre can be identified after 2 months. These finally become the four types of the adult: I, IIA, IIDog, IIC. The main conclusions of the study are that the use of mATPase techniques is appropriate for showing the differentiation between muscle fibres, even at early stages of postnatal development, and that the origin of the four different fibres of the adult can be traced back to early postnatal stages.

Characterization of oral double balloon endoscopy in the dog.

Exploration of the canine small intestine using conventional endoscopy is restricted to the duodenum and/or the ileum. Double balloon endoscopy (DBE) is a 'push and pull' technique that has been described in humans and permits a complete exploration of the small intestine. In this study, oral DBE was performed on 12 healthy dogs (10-34kg) to characterize for the first time the efficiency, exploration dynamics and safety of the technique. DBE was successful in 83% of dogs; the average estimated insertion depth of the endoscope was 287±36cm, and the average duration of the exploration was 84±8min. No complications or relevant adverse clinical effects were observed, and there was no indication of post-procedure pancreatitis based on serology of two specific markers of pancreatitis (amylase and lipase) and the immediate nonspecific inflammatory mediator C-reactive protein. The study showed that oral DBE is viable and safe in the dog, allowing for the diagnosis and treatment of gastrointestinal diseases deep in the small intestine to an extent that has not previously been possible using conventional endoscopy.

Morphometric study of the layers of the canine small intestine at five sampling sites.

The histology of the canine intestine has not been accurately defined. To establish the precise thickness of its different layers, whole wall samples of the small intestine were removed from 41 cadavers at five standardised sampling sites (duodenum, proximal jejunum, distal jejunum, proximal ileum and distal ileum). The total thickness was estimated by morphometry, as was the thickness of the mucosa, muscularis mucosae, submucosa and muscularis externa. In addition, the size of the lymphoid aggregates in the submucosa and the thickness of the circular and longitudinal layers within both the muscularis mucosae and the muscularis externa were estimated. The total intestinal thickness depended very much upon the thickness of the mucosa and submucosa. The mucosa decreased progressively from proximal to distal parts of the small intestine (47% reduction). The thickness of the submucosa, however, changed little from the duodenum to the distal jejunum, but increased significantly in the ileum; this change was positively correlated with the amount of lymphoid tissue. Sex influenced the thickness of the intestinal wall, with males displaying higher thickness values along the small intestine. Conversely, no correlation between bodyweight and intestinal thickness was found for any of the five sampling sites. This study gives absolute and relative values for the thickness of the layers of the dog intestine which might help in the diagnosis of small intestinal pathology from postmortem samples and/or endoscopic biopsies.

Morphometry of the canine intestine with reference to the use of double balloon endoscopy.

Double balloon endoscopy (DBE) enables the diagnosis and treatment of small intestinal disease. The dog is a potential animal model for DBE training and also a candidate for the clinical application of this technique. However, the anatomy of the canine small intestine may limit the use of DBE by restricting the push-and-pull manoeuvres required for DBE. To establish what these limitations are, the length and width of different portions of the small intestine as well as the mesenteric dependences were measured in 55 dogs. Several external parameters related to body size plus the age, weight, sex and breed were recorded. Potential restriction of the progress of the enteroscope due to narrow intestinal diameter is expected in the jejunum of mid- and small size dogs.

Posthatch development of the axial musculature of the common dentex Dentex dentex, L (Teleostei).

The common dentex is a promising candidate for Mediterranean aquaculture. The present work is aimed at describing the development of the axial musculature from hatching to postlarval life. Transmission electron microscopy, histochemical (NADH-TR and mATPase) and immunohistochemical techniques (S-58 and TUNEL) have been used. At hatching superficial red and deep white muscles can be distinguished. Presumptive dermomyotome (external) cells are initially located over the superficial red muscle but shortly (2 days) tend to concentrate towards the epaxial and hipaxial limits of the myotome. Then, these cells enter the myotome and spread around and within the white muscle thus being apparently responsible for the stratified hyperplasia of the myotome. Mosaic hyperplasia is activated during the second half of the larval period and initially relies on differentiation of a population of atypical premyoblastic cells (APC). APC are mononuclear cells with euchromatic nuclei, cytoplasms full of thin longitudinally projected tubules, occasional mitochondria and scattered ribosomes. By the end of the larval period these cells tend to disappear, partly due to apoptosis, but postlarval mosaic hyperplasia continues by differentiation of presumptive myosatellite cells. APC are an unexpected and singular finding of this study which deserves more research, so as to further characterize their ancestry, developmental programme and fate. In addition to the white and superficial red muscle fibres, intermediate (pink) and tonic fibres appear during larval metamorphosis. Later, during the early postlarval life, a new type of slow twitch red muscle fibre is differentiated (red adult type).

Muscle cellularity at cranial and caudal levels of the trunk musculature of commercial size sea bass, Dicentrarchus labrax (Linnaeus, 1758).

In eight specimens of Atlantic sea bass of commercial size (congruent with 350 g) muscle cellularity was studied at two selected sampling levels of the trunk axial musculature: caudal (anal opening) and cranial (fourth radius of the dorsal fin). The following parameters were quantified at both sampling levels: white muscle cross-sectional area, white muscle fibre diameter (900-1200 fibres), muscle fibre number and muscle fibre density. Results showed a higher total cross-sectional area at cranial than at caudal level (P < 0.05), what is related with their different gross morphology. However, the white muscle fibre size distribution, as well as the muscle fibre number and density did not show significant differences between them. This study contributes to typify muscle fibre sampling in sea bass of commercial size what is of great interest for morphometric studies where white muscle cellularity is commonly correlated with textural or organoleptic parameters.

Red muscle development of gilthead sea bream Sparus aurata (L.): structural and ultrastructural morphometry.

The transverse red and white muscle area, the superficial red muscle fibres area and their percentages of mitochondria (%mit), myofibrils (%myof) and sarcoplasm (%sarc) were determined in the Mediterranean teleost gilthead sea bream, Sparus aurata (L.). Fish aged from hatching to 78 days were studied. The proportional growth of the red and white muscles was higher for the red muscle in the first half of the larval stage (1-35 days). Then the opposite relationship was observed. The hypertrophic growth of the superficial muscle fibres was continuous except in the first week after hatching. The percentage of mitochondria and percentage of myofibrils showed a significant change just after the half of the larval stage. Whereas the %mit of the superficial muscle fibres was higher than %myof from the first week after hatching to 35 days (average 66.64%), then the %mit decreased significantly and at 73-78 days both parameters were close to an average value of 50%. The meaning of these morphological changes is discussed in relation to the functional role of the red muscle of larvae and the onset of the gills respiration.