Determining the Best Sectioning Method and Intestinal Segment for Morphometric Analysis in Broilers

Brazilian poultry production is very efficient and demands maximum broiler performance. Therefore, digestive system pathologies have a relevant role. Considering it is difficult to obtain consistent information on intestinal morphometric analysis, this study aimed at establishing essential and clear criteria for the collection of intestinal segments for morphometric analysis. Fifteen 13-d-old broilers were sacrificed and three intestinal segments were collected per bird. Two 3-cm long sections were obtained from each of the intestinal segments. Samples were collected open or closed. The closed samples were transversely, hemicylindrically, or longitudinally sectioned. Samples were processed and stained with hematoxylin and eosin. The number of microscopic fields in each section was counted. Villi presenting the base clearly embedded in the submucosa, no damage or folds, and simple columnar epithelium at the tip were considered adequate for measurements. These villi were counted in each sample. The results shows that hemicylindrical sections presented the highest number of observation fields, with an average of 9.76 fields. Jejunum samples were among the three highest average villi counts, with 18.23 in longitudinal sections and 15.61 in hemicylindrical sections. The results of the present study indicate that hemicylindrical sectioning and jejunal samples were, respectively, the best sectioning method and the best intestinal segment for the morphometric analysis of the intestines of broilers.


IntRoduCtIon
The poultry industry demands maximum performance from broilers.Consequently, digestive system pathologies are extremely relevant, as they may negatively affect broilers' economic potential and, according to Ferreira et al. (2012), increase processing losses due to lack of flock uniformity.
The intestinal mucosa consists of a layer of simple columnar epithelium (Kalil et al., 2000;Luna, 1968) and by villi.These projections of the lamina propria into the intestinal lumen increase nutrient digestion and absorption surface area.Despite the many studies in literature on intestinal morphometry, most do not clearly describe the methodology applied.Relevant data, such as the evaluated intestinal segment, the time between death and sample fixation, how the segments are sectioned, and criteria to measure the microstructures, are not informed (Alvarenga et al., 2004;Fukayama et al.;2005, Okamoto et al., 2009and Shiraishi et al., 2009).On the other hand, Caruso & Demonte (2005), studying the histomorphometry of the small intestine of rats, describe the details of the methodology applied and of the measurement criteria used.The authors describe the time between

Birds
Fifteen 13-day-old Cobb broilers were housed in cages and offered feed and water ad libitum.Birds were sacrificed by neck dislocation, and the intestinal samples were collected immediately after death was confirmed.

Sample collection
In order to obtain samples of adequate size, 3-cm long sections were collected from each intestinal segment (duodenum, jejunum, and ileum).Immediately after samples were cut, the intestinal lumen was washed with 10% buffered formalin in order to remove the lumen content.All samples were placed in individually identified flasks containing 10% buffered formalin.
Each intestinal segment was collected either closed (no exposure of the intestinal mucosa) or opened, with the mucosa exposed by longitudinal incision of the organ from the mesenteric border and then stapled on a paper card measuring 2x3 cm.Samples were stretched and stapled through the mucosa with the serosa placed in contact with the paper card.
Duodenum samples were obtained from two sections: close to the ventriculus (descending part) and close to the jejunum (ascending part).Proximal and distal fragments of the jejunum were collected, considering Meckel's diverticulum as the limit between these two fragments.Ileum samples were collected from the segment located between the ceca.

Collection method
Sectioning method

Determining the Best Sectioning Method and Intestinal Segment for Morphometric Analysis in Broilers
Sectioning of the samples The intestinal samples were sectioned using four different methods.All samples were approximately 1-mm thick.The longitudinal sections (hemicylindrical and longitudinal) were 15-mm long.The sections as described in Table 1.

Sample processing for histology
Samples were dehydrated in graded concentrations of alcohol, cleared in xylene, and embedded in paraffin.Samples were then cut into 4-µm thick sections and stained with hematoxylin-eosin.

Observation field count
Microscopic observation fields were counted under optical microscope (10x magnification) for each type of sample (Figure 5) of each intestinal segment, and the total number of observation fields was duly recorded.

Villus count
Only the villi considered adequate for measurements were counted.A villus was considered adequate when its base was clearly embedded in the submucosa (10x magnification; Figure 6); its body did not present any discontinuity or folds (4x magnification; Figure 7); and simple columnar epithelium was present at the tip (40x magnification; Figure 8).

Statistical analysis
The collected data were submitted to one-way analysis of variance and means were compared by the test of Tukey-Kramer.

RESuLtS
The results of observation field counts and number of villi considered adequate for measurement according to sample type and intestinal segment are shown in Tables 2 and 3.The results presented in Table 2 show that, independently of the intestinal segment (duodenum, jejunum, or ileum), the collection of "closed" samples and the "hemicylindrical" section provided the highest numbers of observation fields per sample.
Table 3 shows that jejunum samples provided higher average counts of villi adequate for measurement, and that samples submitted to "hemicylindrical" section presented two out of the three highest average counts of villi adequate for measurement.

Determining the Best Sectioning Method and Intestinal Segment for Morphometric Analysis in Broilers
for 24 hours, and then transferred them to a medium containing 70% alcohol.
This brief literature review illustrates the diversity of methods applied in histological studies of the intestine.Perhaps the only consistent feature among them is the lack of details on the methods used for sample collection.This was the motivation of the present study, as it was not possible to reproduce the methodology described by those authors.We decided to apply part of the mentioned methodologies and to evaluate some essential aspects, such as the number of observation fields and the number of villi considered adequate for measurement, as well as to clearly define which villus characteristics that are required to determine if the villi are adequate or not.These characteristics were not described in any of the researched studies.Some intestinal samples were collected and the segments opened by the mesenteric border, as described by Okamoto et al. (2009), while other samples were collected without opening, as suggested by Shiriashi et al. (2009).All sections were 1.5cm long and were washed with a fixing solution, according to Fukuyama et al. (2005).
The small intestine samples that were collected and not opened and submitted to hemicylindrical section presented the highest number of observation fields, independently of intestinal segment.This result is in agreement with Kalil et al.(2000), who recommend that small intestine samples should always be sectioned in the longitudinal direction in order to obtain histological section which villi are not unduly increased.
The intestine is a very sensitive organ and needs to be handled with care.In the present study, strict criteria were adopted to consider that a villus was adequate for measurement.The villus body should not present any artifacts (folds, breaks, grooves, etc.), its base should be clearly embedded in the submucosa, and its tip should present simple columnar epithelium.Artifacts in the villus body may "reduce" its size, such as in the case of folds, or increase it, such as in the case of grooves or breaks.A villus base clearly embedded in the submucosa indicates that the villus belongs to the evaluated section, whereas the tip with simple columnar epithelium suggests that it was sectioned close to its sagittal line.The consideration of these characteristics provide higher measurement accuracy, as they prevent possible distortions.When these characteristics were taken into account when evaluating the villi, the samples obtained by hemicylindrical section were shown to be the most adequate for morphological evaluation, because they presented the highest number of microscopic observation fields and the highest number of villi considered adequate for measurement.In addition, because the jejunum presented the highest number of measurable villi and observation field counts, it is suggested that this intestinal segment is the most indicated for morphometric analyses.

ConCLuSIonS
Based on the results obtained in the present study, it was concluded that the samples that were collected closed and submitted to hemicylindrical section presented the highest number of observation fields, independently of intestinal segment.The jejunum is the most indicated intestinal segment for intestinal histomorphometric analysis, because it presented the highest number of villi considered adequate for measurement and due to its importance for nutrient absorption.In addition, it was observed that the structure of the intestines is very frail, requiring extreme care when handled.The present study may be used as the basis of future studies using intestinal morphometric analyses as it elucidated basic issues relative to sample collection method and processing.

Figure 1 -
Figure 1 -Transversal section of the jejunum collected closed Figure 2 -Section of the jejunum collected open and stapled

Figure 5 -
Figure 5 -Diagram of the section surface and of the observation fields of the different types of samples.

Figure 6 -
Figure 6 -Base of a villus considered adequate for measurement (arrow).

Figure 7 -
Figure 7 -Body of a villus considered adequate for measurement (arrow).

Figure 8 -
Figure 8 -Tip of a villus considered adequate for measurement, presenting simple columnar epithelium (arrow).
wide variety of methods for the collection of intestinal samples for histology.For instance,Alvarenga et al. (2004) collected 5-cm rings from each small intestine segment, whereasOkamoto et al. (2009) collected 1-cm long samples of the proximal duodenum that were opened by the mesenteric border.Shiriashi et al. (2009)  collected ring-shaped samples of the ileum and washed them with a 0.9% NaCl solution.Fukayama et al. (2005) collected 1.5-cm long intestinal samples, which were immediately washed with distilled water and fixed in Bouin solution for 24 hours.Caruso & Demonte (2005) collected 1-cm samples of the small intestine of rats, fixed them in 10% formalin, refrigerated the samples Gava MS, Moraes LB, Carvalho D, Chitolina GZ, Fallavena LCB, Moraes HLS, Herpich J, Salle CTP

Table 1 -
Sample collection and sectioning methods applied.

Table 2 -
Average number of observation fields according to sample type and intestinal segment.

Table 3 -
Average number of villi considered adequate for measurement in intestinal samples of 13-d-old broilers.
cMeans followed by different superscripts are significantly different at p≤0.05.