Age-related histomorphological studies on the spleen of Chinese yellow quail

The spleen is one of the most important peripheral lymphoid organs, playing an important role in the immune response to circulate antigens in the blood Guo et al., (2014). The Chinese yellow quail (Coturnix japonica), which is known as a quail-bird or quail, is one of the excellent varieties of quails both at home and abroad. At present, studies on quail mainly focus on production performance (Johri et al., 1990 and Ayasan 2013). The quail spleen has also been studied, but research on it was focused on specific age groups. For example, some scholars studied the structure of an 8-week-old spleen (Wang et al., 2009). Others studied the spleen and quail embryos between 0 and 16 days before embryonic tissue was formed and then between 1 and 60 days after embryonic tissue structure was formed (Liman et al., 2011). With the rapid development of poultry breeding industry, poultry disease has become a thorny issue (He et al., 2016). However, the study of the entire development of the structure of the spleen has not been reported, which prompted this present study.

Animals
 
A total of ninety Chinese yellow quail were procured from Pengdingchun farm (Qionglai, China). The quails were randomly divided into 15 groups (6 in each group) at the ages of 0, 1, 2, 3, 4, 5, 6, 10, 14, 18, 22, 26, 30, 34 and 38 weeks. All the birds were treated in accordance with the Guidelines for Care and Use of Laboratory Animals and the study was approved by the Animal Ethics Committee of Sichuan Agriculture University.

The quails were anesthetized with an intraperitoneal injection of 10% chloral hydrate (2 ml/kg) and placed on an electronic balance to obtain the body weight, which was then recorded. The sternum of the quails was then incised at the midline to expose and collect the spleen.

After thorough cleaning, the spleen was weighed by using an electronic balance. The organ index was calculated using the formula:
 

Organ index= (Organ weight/Body weight) ×100%.

 
Finally, the length and width of the spleen were measured by using Vernier Calipers.
        
The spleen samples were collected at different ages and fixed in 10% neutral buffered formalin sections of 5-6 µm were obtained using paraffin method and subsequently stained using haematoxylin and eosin (Drury et al., 1980) staining to study its histomorphology.The average area and average diameter of the splenic nodules, ellipsoid and periarterial lymphatic sheath of the spleen were then measured at a magnification of 100× using Jiangsu Jetta image analysis software. Data were expressed as the mean ± standard deviation (c ±s). All statistical analyses were performed using one-way ANOVAs using SPSS 22.0 software.

The spleen of the Chinese yellow quail was located on the right dorsal side of the junction of the muscle and stomach and the glandular stomach (Fig 1a). It is brownish red in colour and spherical in shape (Fig 1b).



The weight of the Chinese yellow quail increased with age from 0 to 14 weeks, at which point the weight reached a peak (156.5833 mg).The absolute weight increased with age from 0 to 6 weeks and it reached a peak at 6 weeks (104.3333 mg). The organ index reached a peak at 6 weeks (0.7585).The weight of the the Chinese yellow quail, the absolute weight of the spleen and the organ index was significantly greater at 6 weeks of age than at 0 to 5 weeks (P<0.01) and there was no significant difference compared to at 14 to 38 weeks (P>0.05).

The spleen was composed of a capsule and parenchyma. The capsule, which is thicker, is made up of connective tissue and smooth muscle and extended into the parenchyma to form the underdeveloped splenic trabeculae. The parenchyma was divided into the white pulp, the red pulp and the marginal zone. The boundaries of the white pulp and red pulp were clear and staggered (Fig 2b).

The white pulp consisted of dense lymphoid tissue around the artery, including the periarterial lymphatic sheath, splenic nodules and ellipsoids (Fig 2b). Sometimes, there were diffuse lymphoid tissues outside of the ellipsoid (sheath and artery) and these lymphoid tissues showed different shapes, such as petal (Fig 2f), round (Fig 2c), oval (Fig 2i) and long strip (Fig 2d). The red pulp, which included splenic cords and splenic sinuses, was located around the white pulp, and both the red and white pulp were interlaced with each other (Fig 2b).

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The volume of the spleen gradually increased with age, reaching a peak at 14 wk of age and then remained at a certain level after 14 wk. The average area and average diameter of the splenic nodules, the periarterial lymphatic sheath and the ellipsoid increased up to a certain age and then remained approximately the same. At 0 weeks of age, the number of lymph nodes was greater, there wre more dense diffuse lymphoid tissues, and the ellipsoid embryonic form had appeared. The reticular cells revolved around the formation of the group of lymph nodes, forming the initial splenic nodules. At the same time, there were a large number of vessels forming in the spleen, as well as a few vessels that already formed (Fig 2a). The basic shape of spleen nodules appeared on 1~2 weeks’ age. The splenic nodules with clear outline and complete structure could be observed at 3-4 weeks of age. The cells in the splenic nodules were arranged closely, and the boundaries of the surrounding cells were not clear, and the ellipsoid structure was typical (Fig 2e). At 6 weeks of age, the outline of the splenic nodules in the white pulp was clear. The circumference of the peripheral cells was covered by one or two layers of reticular cells, clearly defined with the surrounding cells, the splenic nodules were common, there were more lymphatic sheath around the arteriesThe number of ellipsoids was more, the volume of the ellipsoid was larger and the volume of the sheath was visible in many ellipsoids and the volume of the ellipsoid was less (Fig 2g). At the age of 10-26 weeks, the spleen did not change in its architexture, the volume of the splenic nodules increased, the area of the lymphatic sheath around the artery increased, the volume of the ellipsoid increased, the number increased and the red and white pulp boundaries were clear and interlaced (Fig 2h-l). At the age of 30-38 weeks, the volume of the splenic nodule, the area around the lymphatic sheath and the volume of the ellipsoid decreased gradually (Fig 2m-o).

There are obvious differences in the morphological characteristics of the spleen among poultry such as ducks, geese and pigeons, which have a long and triangular spleen (Chen 2002). In this study, the position of the Chinese yellow quail spleen was located between the gizzard and proventriculus at the junction of the right dorsal, similar to other birds and its morphology is similar to that of the chicken, being spherical and reddish brown. The results showed that the Chinese yellow quail spleen had already formed prior to the quail hatching, and from 0-38 weeks of age, it had a complete external form. The surface was smooth and plump and the ratio of length to width (approximately 0.75) changes little with age, showing that the spleen had a degree of stability in the appearance and essence of its structure.

The dynamic observation of the macroscopic growth index of the spleen is an important basis for measuring the normal growth and pathological condition of the spleen. The present study revealed that the weight of the quail, the weight of the spleen and the organ index were increased with age. The weight of the the Chinese yellow quail, the absolute weight of the spleen and the organ index were significantly greater at 6 weeks of age than at 0 to 5 weeks, indicating that the spleen developed rapidly at these stages, and there was no significant difference compared to at 10 to 38 weeks, suggesting that the development of the spleen remained dynamic and stable between the ages of 10 and 38 weeks, all of this indicating that the spleen development occurs between 0~6 weeks and then matures from 10~38 weeks. It can be seen that the spleen had a phased, rather than linear, growth (Cheng et al., 2007). Thus, the changes in the index and absolute weight showed a similar trend to that found by Chen (2011) and Shu (2009), indicating consistent results among different studies.

This research showed that the spleens of quails developed late. At the age of 0-1 weeks, the blood vessels were clear and distinguishable. Most of the other lymphocytes were dense, diffuse lymphocytes, and there were only a few formed lymphatic sheaths. This finding is similar to that of Wang et al., (2009). During this period (0~1 weeks old), the present study indicated that the spleen of the quail had not yet formed a complete and effective tissue structure for immune response. The structure of the splenic nodules was clear at the age of 3 weeks, indicating that the spleen was forming its immune basis at this time, and the spleen was gradually transformed into the lymphoid organ as a hematopoietic organ. The average area and diameter of the splenic nodules, periarterial lymphatic sheath and ellipsoid in the spleen of 3-6-week-old quails gradually increased, indicating that the spleen development was more active during this period and had begun to participate in the histological basis of immune response. Although the routine morphological indexes of the spleen remained basically unchanged, the average area and diameter of the splenic nodules, periarterial lymphatic sheath and ellipsoid increased with age from 10-26 weeks of age. This finding indicated that during the duration of spleen maturation, its immune function was gradually increasing, which showed a slight decrease at 30-38 weeks of age, indicating that the immune function of the spleen was not as strong as before.

The B lymphocytes in the spleen are mainly derived from the central immune organs (Kong et al., 2002). B lymphocytes are the first targets in the central immune organs, and the surviving B lymphocytes that migrate to the peripheral immune organs after further differentiation. The mature T lymphocytes migrate out of the thymus and into the peripheral lymphoid organs (Jeurissen, 1993). The spleen has a distinct T and B lymphocytes, and the structure of the spleen is closely related to the central immune organs (Kumar et al., 2007). The bursa of the quail reached a maximum size at the age of 39 days and then gradually degenerated (He et al., 2011). However, the quail spleen reached maturity at 6 weeks of age and was then mature and stable. It showed that the immune system of the quail was mainly reliant on the spleen after reaching sexual maturity.

The differences and correlations between the morphological characteristics and histological structure of the quail spleen and discussion of functional strength can provide a scientific theoretical basis for disease prevention, timely immunization, clinical diagnosis and disease control.