Whole mitochondrial DNA (mtDNA) S. plagiostomus was 16569bp with 2 rRNAs genes, 22 tRNA genes,13 proteincoding genes, and 2 non-coding region. For estimating association of phylogenetic of a species based on Protein coding mitochondrial DNA genes are measured to be a great value. We sequenced protein-coding genes of Schizothorax
plagiostomus in present study, based upon these genes we compared with S. plagiostomus the 48 Schizothoracinae fishes by phylogenetic association. We analysed that S. plagiostomus to be closely related with Schizopyge niger, Schizothorax labiatus, Schizothorax nepalensis with high-bootstraps values. For understanding phylogenetic relationship of Schizothorax species such empirical data would be important.
Gender-comparison; Histology; Japanese quail; Lymphoid organs; Morphology
The poultry industry day by day improving the meat and egg productive capacity of chickens and Japanese quail to fulfill the requirement of high-quality proteins [1,2]. The poultry industrial part has much practiced in dynamic growth. Poultry production rate has been improved; it contributes total livestock production which is growing quicker production. Poultry growth has been accompanied by huge changes in the structure of poultry production . Although, regarding Japanese quail histomorphology, it is yet not to be done. Therefore, the present research has been carried out to understand the histomorphological architecture of the lymphoid organs. Japanese quail have relatively early maturity and used as an experimental model because of their rapid development and small size .
The Japanese quail have a short generation interval, which having the capacity to produce 3-4 generations per year . The lymphatic system is a defense mechanism and provides a basic immunity [6,7]. The Avian thymus primary lymphoid organ undertake age dependent changes which are alarm and fragmentry that cause structurally and functionaly involution in thymus. As the age increase quail thymus shows a clear difference between the cortex and medulla. Age plays very important role in involution changes which are caused by proliferation of connective tissue fiber, propagation of connective tissue and their muscle cells, it can be observed at about 5-6 weeks in quail .
The spleen is an important peripheral lymphatic organ and the site for immune response production . Splenic central artery is divided into small central arteries that possess one muscle layer and several minute capillaries which are enclosed by sheaths . The pulp is the part of the circulation; chicken spleen has an opened circulatory arrangement . Previous study showed that the light and electron microscopic studies show that the capillaries which enter the red pulp are continuous with the sinuses and gradually take on their structure . The red pulp of the spleen is composed of connective tissue known as the cords of Bill Roth and many splenic sinuses that are enlarged with blood, giving it a red color. White pulp is formed by lymphatic tissue, Branches of the splenic artery which divides into trabecular arteries .
The bursa of Fabricius is distinctive and peculiar organ which provides microenvironment for maturation, differentiation and grants immunity of bird but also to the T lymphocyte cells [13-16]. It consists of some blind, crypt-like invaginations (plicae) functionally organized in bursal follicles that contain heterogeneous cell populations, mainly lymphocytes, epithelial and secretory dendritic cells. Secretory dendritic cells obtained from the mesoderm and limited to a small area in the medulla of the follicles [17-19]. The secretory dendritic macrophage-cells are dispersed throughout the lymphoid organ .
Avian large intestine consists of paired caeca and a short straight colon joined to ileum and cloaca. Quails intestinal villi whose feature according to Naik as long their arrangement that are histologically similar to the small intestines [21,22]. The mucous membrane is similar to that of the small intestine, with less goblet cell and fewer glands. The villi are well developed at the basilar part, shorter and wider in the middle part and either shorter or absent in the apex . As far as we know, the effect of age on the histological parameters of quails villi has not been evaluated yet. This may be important for better clarification of the absorptive and immunologic capacity of villi at different ages. Considering the importance of the lymphatic system the results of the presentstudy will provide some basic data on the morphogenesis and development of the lymphoid organs and aimed to achieve age-related changes in shape, size and histomorphology of thymus, spleen, bursa of Fabricius and villi in Japanese quail.
Twenty-four chicks of different age groups were purchased from a commercial hatchery (Animal Husbandry Co. Ltd Karachi Pakistan) after overnight fasting quail chicks were offered ad libitum diet and Animal experiment was conducted after approval of institutional animal welfare and research committee Sindh agriculture university Tando Jam Pakistan, quails chicks were randomly divided into three age groups, i.e., A (2 weeks), B (4 weeks) and C (6 weeks), each group was consisting of 8 birds (4 males and 4 females). Eight birds from each group were sacrificed by cervical dislocation under ether anesthesia on commencement of the experiment.
Morphological analysis and sample collection
Quail chicks were slaughtered and removed the whole intestinal tract safely, afterward the small intestine (Duodenum, jejunum, ileum) and large intestine parts were cut with the help of scissor and feces were washed with the help of normal saline. A gross anatomical study was done immediately after collection of lymphoid organs (Thymus, spleen, bursa of fabricius and intestinal villi) according to a study . The weights of organs were measured with an electrical balance, length of the thymus with measuring scale from cranial lobe to caudal lobe. Whereas; width of the thymus was measured dorso-ventrally using digital vernier caliper . The length of bursa of Fabricius and spleen were measured from the cranial pole to caudal pole using a measuring scale. The width of bursa fabricius and spleen were measured from lateral border to medial border as stated earlier .
Hematoxylin and Eosin (H&E) staining
Histological study was performed on 3 μm thick transverse section, cut by microtome then fixed on slide and stained with haematoxylin and eosin, counter stain then tissue sections were examined on a Nikon phase contrast microscope coupled with a Microcomputer integrated digital imaging analysis system (Nikon Eclipse 80i, Nikon Co., and Tokyo, Japan).Lymphoid tissues thymus, spleen, bursa Fabricius histologicaly measured and Villus height was measured from tip (with a lamina propria) of the villus to the base (villus-crypt junction) and villus width was measured at its middle part .
Gross anatomical parameters of major lymphoid organs were computed by using Microsoft Excel Software. Group means of length, width and weight of major lymphoid organs were compared in a one-way analysis of variance (ANOVA) with (Tukey’s Kramer software 7.0 Version) and significant differences were compared through Student’s comparison test.
Ethical approval and consent to participate
This study was approved by the Institutional Review Sindh Agriculture University Tando Jam Pakistan. However the validity of conventional possession owner was completely assured in proceed our data
The Morphohistological studies were performed on major lymphoid organs; thymus, spleen, bursa of Fabricius and intestinal villi of a healthy male, female quails of different ages.
Morphology of lymphoid organs
Thymus: The Avian thymus is considered as a primary lymphoid organ. The color of the thymus was observed light (pale) white to yellowish white, and the shape of the lobes was typically extended as well as obviously flattened in Japanese quail.
The weight, length, and width of right and left thymus of all three age groups were insignificantly (p>0.05) slightly higher in females than males (Table 1). Whereas, significantly (p<0.05) higher in group C, B and A respectively (Table 2).
|Weight (gm)||Length (cm)||Width (cm)|
|Weight (gm)||Length (cm)||Width (cm)||Weight (gm)||Length (cm)||Width (cm)|
Table 1: Sex-wise morphological differences of thymus, spleen and bursa in Japanese Quails on different ages 2W=2 weeks, 4W=4 weeks, 6W=6 weeks, L/R=left/right, P.V=probability value (p≤ 0.05), gm=gram, cm=centimeter.
|Groups||Weight (gm)||Length (cm)||Width (cm)|
|Groups||Weight (gm)||Length (cm)||Width (cm)||Weight (gm)||Length (cm)||Width (cm)|
|A||0.054c||0.241 c||0.150 c||0.101 c||1.070 c||0.093 c|
|B||0.137b||0.371 b||0.287 b||0.205 b||1.190 b||1.190 b|
|C||0.235a||0.445 a||0.375 a||0.317 a||1.326 a||0.315 a|
Table 2: Age-wise morphological differences of thymus, spleen and bursa in male and female Japanese Quails. A=2 weeks, B=4 weeks, C=6 weeks, L/R=left/right, P. V=Probability Value (p ≤ 0.05), gm=gram, cm=centimeter.
Spleen: Morphologically the quail spleen seen reddish brown color and slightly rounded in shape enclosed by a dense capsule and number of trabeculae. The weight, length, and width of the spleenwas insignificantly (p>0.05) higher in females than the males, whereas it was observed significantly higher (p<0.05) in group C, B and A respectively (Table 1).
The absolute weight, width, and length of the spleenwere observed high in group C, B and A respectively (Table 2).
Bursa of fabricius: The lymphoid organ bursa fabricius evaluated as light pinkish in color, blind, globular and oval shape in Japanese quail. The weight, length, and width of bursa Fabricius was observed insignificantly (p>0.05) higher in females as compared to males. Furthermore, it was observed significantly (p<0.05) higher in group C, B and A respectively (Table 1).
The absolute weight, width, and length of bursa Fabricius was examined high in group C, B and A respectively (Table 2).
Intestinal villi: Avian small intestine consists of duodenum and a short straight jejunum joined to ileum and cloaca. Quail intestinal villi are small in size, finger like in shape that extends form space of lumen in small intestine, villi sac consists of 3 parts: proximal part or base, middle and distal part or apex. The quail villi are well developed at the basilar part, shorter and wider in the middle part and either shorter or absent in the apex.
Histological studies of lymphoid organs
Thymus: Thymus undergo the various developmental stages on the base of their sex, it shows underdeveloped fine fibrous connective tissue capsule, incomplete septa which were invaded by lobules into dense cortex and light medulla during 2nd week of thymus that can be observed in group A. Such changes enhance as age grown when it reached at 4th week, quail thymus shown developed fine fibrous connective tissue capsule and complete septa that divided into lobules in distinct cortex and light medulla, these changes rely in group B and sequencely age rechead at 6th week thymus showed well-developed lobules with the prominent peripheral cortex and central medulla.Prominent appearance may be lost due to the decreased number of cortical, medullary epithelial cells and Hassall’s corpuscles such examination can be in observed group C (Figure 1).
Figure 1: Histological measurements of villus length and width. (A) Representing sex wise differences. (B) Representing group wise differences. M=male, F=female. 2 Wks=2 weeks, 4 Wks=4 weeks, 6 Wks=6 weeks. H=height, W=width. Inset A, B, C=2 weeks, 4 weeks and 6 weeks respectively. Superscripts a, b, c are significantly higher. Measurement unit is µm.
Spleen: Spleen primarily blood filter organ on the base of their sex, it was encapsulated by thin tissues of fibroblastic conatined numerous smooth muscle cells and elastic fibers in their deeper parts of elliptical capsule, on the base of their sex due to parsing trabecule parenchymatic cell could not be visible in smaller compartment during their underdeveloped stage. As age grow from underdevloped to advanced stage differentially types of splenic nodules were easily visibles. It can be differentiated by the presence and absence of germinal center, moreover red and white pulp mean length observed insignificantly (P>0.05) higher in females than the males shown in Table 3, whereas, it was observed significantly (P<0.05) higher in group C, B and A respectively (Table 4 and Figure 1).
|Age||Length of lobules (µm)||Width of lobules (µm)|
|Age||Length of white pulp (µm)||Width of white pulp (µm)|
|Age||Length of follicles (µm)||Width of follicles (µm)|
Table 3: Sex-wise histological differences of thymus, spleen and bursa in Japanese Quails on different ages. 2W=2 weeks, 4W=4 weeks, 6W=6 weeks, L/R=left/right, P. V=probability value (p ≤ 0.05), µm=micrometer.
|Length (µm)||Width (µm)||Length (µm)||Width (µm)||Length (µm)||Width (µm)|
Table 4: Age-wise histological differences of thymus, spleen and bursa in male and female Japanese Quails. A=2 weeks, B=4 weeks, C=6 weeks, L/R=left/right, P. V=probability value (p ≤ 0.05), µm=micrometer.
Bursa of fabricius: Bursa of Fabricius specialized lymphoid organ, it contained primary lymph tissue which made up of B lymphocytes cells that participate in humoral immunity response, on the base of sex bursa made up of follicles which are separated by fine fibrous connective tissue obvoiusly it can be observed in peripheral cortex and central medulla, follicles are less visible in 4 and 6 weeks quail, degenration may occurred in follicles due to loss of their interstitial fibrosis and follicles such evidence are much visible during their enhancement of age. Furthermore bursal follicles mean length and width was observed insignificantly (P>0.05) higher in females then male quail as shown in Table 3, whereas it was observed significantly (p<0.05) higher in group C, B and A respectively in Table 4 and Figure 2.
Figure 2: Histological changes of thymus, spleen and bursa (A) Representing thymus. (B) Representing spleen. (C) Representing bursa. C: Capsule, Crt: Cortex, M: Medulla, HC: Hassall’s corpuscles, RP: Red pulp, WP: White pulp, F: Follicles. Bar=100.
Intestinal villi: Histological examination revealed that in quail the finger-like projections villi extend form lumen of small intestine that enclosed by simple columnar epithelium which composed of four layers, i.e., tunica mucosa, sub mucosa, muscularis,and serosa. It generates goblet cell arranged and scattered in lymphoid tissue which secretes mucin, whereas in intestinal villi could not find any sex difference in height and width of the simple columnar tissues. Furthermore, its height and width were higher followed by bursal follicle, thymic lobules, and splenic red and white pulp.
The avian immune system provides a significant experimental model for studies on basic avian immunity. The lymphoid organs play an important role in body defense system, maintain normal function of immunity and promote resistance against the disease in birds and animal as well. However, the thymus primary lymphoid organ in quails was light (pale) white to yellowish white this similarities accordance to in duckling and the shape of the lobes was typically extended as well as obviously flattened, located in parallel with the jugular veins and the vagus nerve in both sides of the neck region. These outcomes regarding their location of the thymus are arranged by King and Mc Lelland in fowl and quail, and by Raymond et al., and Akhter et al., 2006 in chicken, in turkeys [26-28]. The quail thymus contained thymic lobes which extend to 7-8 on the right side and 6-7 on the left side. These all dissimilarities regarding to thymus lobes might be differed due to species difference. The timely process of involution, arrangement, and functions of the lymphoid organs were greatly affected on depletion of lymphocyte and plasma cell which cause structural changes on thymus such changes may cause a decline in thymus weight respectively, Such findings are similar to the finding made by Baumgartner, Ebru karadag et al., David Huss et al., and Haseeb et al. This histomorphological examination also observed in turkey, Duckling, Aseel chicken and fowl as well [1,29-31].
Quail primarily lymphoid organ thymus contained elliptical lobules which are separated by numerous fine, tinny fibrous connective tissue around abundance dense capsule which are existed nearby fine slightly irregular fibrous connective tissue in peripheral cortex and central medulla, such histological similarity are according to a study . However, age of avian reach to their peak thymus become involuted and reduce intheir size this fact similar to some studeis [8,17]. Furthermore, as the improvement of age thymus reduces their weight due to depletion of lymphocyte, it can be observed at 5 to 6 weeks of age in male and 6 to 7 in the female. This observation accordance with , a significant difference (P<0.005) was found in the length and width of thymic lobules among three age groups. As the quail age increased regression of thymic lobules occurs and clear distinction between medulla and cortex may be observed, this fact is close correspondence with a study .
Morphological characteristic of spleen shown that The quail spleen seen reddish brown color and slightly rounded in shape enclosed by a dense capsule spleen and number of trabeculae closely located to the right side of the tightly junction between proventriculus and the gizzard such characteristics of spleen is in correspondence with [28,34]. Spleen is made up of significant cell parenchyma contained red pulp and white pulp, red pulp these similarities accordance to Indu et al., as spleen enhance in their size by the advancement of age, the weight, length and width of spleenwas insignificantly (P>0.05) higher in females than the males , whereas, it was observed significantly (P<0.05) higher in group C, B and A respectively, this conclusion correspondence with .
Quail spleen encapsulated by numerous thin fibroblastic tissues which are made up of parenchyma, smooth muscle cells and elastic fibers, in their underdeveloped age parenchymatic cells of spleen could not be visible in tinny compartment of spleen due to parsing trabeculae these similaity according to Akhter et al., Spleen tinny cells parenchyma contained obviously network of reticular cells and connective tissue, this difference between red and white pulp disappear as their age grow, it can be observed by presence and absence of germinal center such Similar findings are according to Akter et al. as their germinal center disappear the trabeculae become less prominent this conclusion corresponding to studies [28,34]. Moreover, Statistical analysis showed that the mean length and width of white pulp insignificantly (P<0.05) in both sexes and among age groups because of sex correlated.These results could be compared with Venkatesan et al., Akter et al. and Haseeb et al., in Fowl and Aseel chicken [24,28,34,35].
The bursa of Fabricius in Japanese quail is an immunological organ that playsa primitive role in poultry immunity; it placed in the dorsal diverticulum proctodeal wall of entrained in the cloaca. The bursa of Fabricius in Japanese quail was light pinkish in color, blind, globular and oval shape such characteristics are also observed in chicken and ostrich as well, and these observations are in accordance with [28,36,37].
The histological evaluation shown that the wall of bursa of Fabricius in Japanese quail contained three main layers 1st tunica mucosa, 2nd tunica muscularis,and 3rd tunica serosa, the tunica mucosa formed from clear plicae with different width and length and Plicae outer layer surrounded by pseudostratified epithelium these lines are given similarities with Yamada et al., such similarities accordance to [8,31,36]. It has also been histological examination shown that; the regressions of bursal follicles occurred with the improvement of age. Regression Changes in quail bursa are rare, heterogeneous and fragmented and a clear difference was observed between medulla and Cortex in Gallus domesticuswhich were lost with aging. This conclusion is correspondence to previous studies [36,38]. As the advancement of age occurred bursal follicles showed involutive changes due to atrophy of plicae epithelium in which epithelial fibrosis, plicae medullary and cortical cells are collapse. Moreover mean weight, length and width in bursa was observed insignificantly (P>0.05) higher in females as compare to males, however it was significantly (P<0.05) higher in C, B and A respectively this similarity according to accordance to previous studies .
In this present study. It was observed that the intestinal villi the finger-like projections which expand into the one-third of the intestinal tubular duodenum of the small intestine. It composed of four histological layers: i.e., tunica mucosa, sub mucosa, muscularis,and serosa. In the earlierstage, the wall of villus was made up of simple columnar epithelium which contained enterocyte and goblet cell. Enterocyte which increases their surface area to enhance the diffusion, whereas, goblet cell which was scattered in lymphoid tissue secrete mucin this statement shows similarities with . Furthermore, its height and width were higher followed by bursal follicle, thymic lobules and splenic red and white pulp such characteristic gives similarities with .
It is concluded that considerable changes that rely on age and sex dependent the size of thymus, spleen and bursa of Japanese quail increased to its max at 6th week of age and after that it started to reduce in size. There was no significant difference in lymphoid organs of Japanese quail with respect to sex. A positive increasing pattern was observed for weight, length, width and diameter of lymphoid organs from 2 to 6 weeks. Our findings contribute in basic data on the growth dynamics in histomorphology of the lymphoid organs. Further studies are suggested to examine ultrastructure and different immune histo-chemical techniques of lymphoid organs from pre-natal to puberty stage to understand developmental changes at cellular ultra-structural level with respect to the cell shape and size during the course of growth.
Authors declare that there is no conflict of interest.
Mangi RA, designed the Study, collect data, prepared instrument, analyzed data and write-up manuscript, M.G.S supervised data collection & data analyzed, A.R.J help in data analyzing and manuscript write-up, P.A.K and K.N help in data analyzed, M.H.M, Afrasyab help in data collection.
The work was supported by self with moral help of Department of Veterinary Anatomy and Histology Sindh Agriculture University Tando jam Pakistan. All associated authors have read carefully and approved the final manuscript.
The study was conducted at Sindh Agriculture University Tando Jam Pakistan. We authors are thank full to Department Anatomy and Histology/Veterinary Medicine for providing lab faculty and experimental facility.
Citation: Bibi S, Khan MF, Rehman A, Nouroz F (2019) Polyphyletic Origins of Schizothoracinae Fishes (Cyprinidae) in Respect to Their Mitochondrial Protein-Coding Genes. Poult Fish Wildl Sci 7:207. doi: 10.35248/2375-446X.19.7.207
Received Date: Sep 03, 2019 / Accepted Date: Oct 24, 2019 / Published Date: Oct 31, 2019
Copyright: Â© 2019 Bibi S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.