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Histological assessments of intestinal immuno-morphology of tiger grouper juvenile, Epinephelus fuscoguttatus

Histological assessments of intestinal immuno-morphology of tiger grouper juvenile, Epinephelus... Histological assessments on the intestinal morphology and immunity of tiger grouper juveniles, Epinephelus fuscoguttatus help in determining the earliest age to start an oral vaccination. This study describes the morphological development of the intestinal immunity of tiger grouper of various ages. Clinically healthy tiger groupers were selected and divided into 4 groups of 20 fish per group. Groups 1, 2, 3 and 4 consisted of juveniles of 30, 60, 90 and 120 days old, respectively. The whole intestine was collected and divided into three regions, the anterior, mid and posterior intestine and fixed in 10% buffered formalin before slides were prepared for microscopic examinations. It was found that the histological structures of the anterior intestine were for absorption of nutrient from digested food particles. The significantly (p < 0.05) higher number and length of the intestinal villi and smaller gap between villi were observed in the anterior intestine, which were structures for absorption. Structures of the posterior intestine were for immunity especially the adaptive immunity with included significantly (p < 0.05) higher numbers of the lymphoid and goblet cells, and significantly (p < 0.05) thicker lamina propria, which were structures for immunity. The mid intestine was the transition structure that involved in both absorption and innate immunity. The results also revealed that leukocytes existed in the lamina propria of 30-days old tiger groupers, an indication that the immune system was present at that particular age. Keywords: Tiger grouper; Intestine histological; Assessment; Immune system Introduction artisanal fisheries, which groupers are the major compo- Grouper is a high value cultured marine fish especially in nent. According to Food and Agriculture Organization the Southeast Asia countries including Malaysia, Indonesia, (FAO) (2013), world aqu of (2880saculture production of Thailand and the Philippines (Liao et al., 2001; Yashiro, groupers was around 6000–7000 tones per annum, valued 2008). The major cultured species are giant grouper, Epine- at about USD60 million and the bulk of this production phelus lanceolatus (Yashiro, 2008), tiger grouper, Epinephe- came from wild seed stock due to lack of reared seed that lus fuscoguttatus (Sugama et al., 2008), malabar grouper, suffered disease problems. Epinephelus malabaricus (Yashiro, 2008), orange spotted In Malaysia, tiger grouper, Epinephelus fuscoguttatus, grouper, Epinephelus coioides (Toledo et al. 1996), hump- also known as brown marbled grouper, is a major cul- back grouper, Cromileptes altivelis (Marte, 2003), leopard tured marine species. It is considered a popular and coralgrouper, Plectropomus leopardus (De Silva, 1998) and high valued aquaculture species and fast growing (Afero hybrid grouper, (Epinephelus lanceolatus X Epinephelus et al., 2009). Data from Federal Agricultural Marketing fuscoguttatus). Heemstra and Randall (1993) estimated that Authority of Malaysia (2013) (FAMA) indicated that 90% of the world’s harvest on marine food is derived from grouper species are the major export fish for the year 2010 (MAHA, 2013). Fish digestive system is slightly differ from mammal. The * Correspondence: zamri@vet.upm.edu.my Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor digestive process has started in the first region that in- 43400, Malaysia cludes the mouth, oral cavity, and pharynx. Then, after Full list of author information is available at the end of the article © 2013 Firdaus-Nawi et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 2 of 13 http://www.springerplus.com/content/2/1/611 food is swallowed it will enter the alimentary canal proper by an overdose of Ethyl 3-aminobenzoate methanesulfo- and proceeds via the esophagus to the stomach followed nate (Sigma Aldrich, USA). Post-mortem examination by the intestines (Buddington and Kuz’mina, 2000). The was performed immediately; the entire intestine was re- main function of fish intestine is to complete the digestive moved and was cut into the anterior, mid and posterior process, which started in the stomach and also to absorb portions (Wilson and Castro, 2011) before the intestinal the nutrients from food (Wilson and Castro, 2011). Other samples were fixed into 10% buffered formaldehyde for than the digestive function, gut of fish also acts as the first- at least 24 h. The Animal Care and Use Committee of line barrier against infection. Thus, the mucosal layer of University Putra Malaysia approved the study protocol. the gut creates physical, chemical and cellular protections against pathogen invasions (Ellis, 2001). The goblet cells Sample preparation and histological analysis and the glandular simple columnar epithelial cells se- Following 24 h fixation, the samples were prepared for crete mucus, which contains immunological substances histological examination according to Firdaus-Nawi et al. such as glycoprotein (Fletcher and Grant, 1968), cyto- (2012). The slides were examined under a light micros- kines (Lindenstrøm et al. 2003), peptides (Cole et al., copy using Nikon NIS-Element D 3.2 Image Analyzer 1997), lysozyme (Fernandes et al., 2004), lipoprotein (Nikon Instruments Inc., USA). For each slide, a total of (Concha et al., 2003), complement (Dalmo et al., 1997), five microscopic fields were examined at 200× magnifica- lectins (Tsutsui et al., 2005), proteases (Aranishi and Mano, tion to determine the number of villi, the length of villi, 2000) and antibodies (Cain et al., 2000). These substances the gap between villi, the thickness of lamina propria, the provide direct or indirect protection against pathogen number of lymphoid cells, the number of goblet cells and (Cain et al., 1996). Furthermore, the intestine of fish con- the thickness of muscular layer. The Nikon NIS-Element tains lymphoid cells that secrete antibodies and involve in D 3.2 Image Analyzer (Nikon Instruments Inc., USA) was phagocytosis. The aim of this study is to describe the histo- used to measure all parameters. logical evaluation on the intestinal immune-morphology of tiger grouper, Epinephelus fuscoguttatus juveniles. Statistical analysis The Statistix 9 (Analytical Software, USA) was employed Materials and methods to analyze the data. The results revealed significance in Fish and rearing conditions all pairwise comparison under one-way ANOVA. Signifi- A total of 80 healthy tiger grouper (Epeniphelus fuscogutta- cant differences were determined at P < 0.05. Pearson tus) juveniles of different ages were used. They were 30, 60, correlation test was used to reveal the correlation be- 90 and 120 days old of approximately 1 g ± 0.3 g, 2.5 g ± tween each studied parameters. 0.3 g, 200 g ± 0.3 g and 300 g ± 0.3 g body weight, and of approximately 2.5 cm ± 0.5 cm, 5 cm ± 0.5 cm, 7.5 cm ± Results 0.5 cm and 10 cm ± 0.5 cm in length, respectively. The se- Number of villi lected juveniles were grouped according to the age with 20 There were significant (p < 0.05) differences in the numbers fish per group and kept separately in 100-L glass tanks. of villi in the three regions of intestine (Figure 1). The aver- Fish of less than 30 days old were too small for handling age number of villi of anterior intestine was 39.200 ± and sampling. All the fish were acclimatized for at least 6.1435, 64.467 ± 11.077, 74.333 ± 11.709 and 95.533 ± 7 days prior to experiment. Light cycle was held constant 4.0860 for tiger groupers at 30, 60, 90 and 120 days old, with 12 hours of lighting per day. Feeding was ad libitum respectively. The average number of villi of mid intestine with a local commercial feed while water was continuously was 22.533 ± 3.6227, 49.933 ± 2.8149, 57.867 ± 6.8334 and aerated. The water temperature, pH, salinity and dissolved 62.000 ± 8.000 μm at 30, 60, 90 and 120 days old, re- oxygen were measured daily using the HQ40d Meter spectively. The average number of villi of posterior in- (Hach Company, Loveland, CO). The ammonia, sulfate testine was 29.067 ± 5.1195, 43.333 ± 4.7006, 44.533 ± and nitrites were determined daily using the DR 2800 Port- 3.3989 and 51.467 ± 4.8236 μm, respectively. able Spectrophotometer (Hach Company, Loveland, CO). The number of villi was highest in the anterior region Prior to sampling, five fish from each age group were sacri- of intestine (Figure 2), followed by the mid region and ficed and swab samples from the organs were collected for least in the posterior region, except for the 30 days old bacterial and parasitic examinations. This was to ensure tiger grouper that showed that the number of villi was that the fish were free from bacterial and parasitic diseases significantly (p < 0.05) higher in the posterior than the (Firdaus-Nawi et al., 2012). mid region. In general, the number of villi showed grad- ual and significant (p < 0.05) increase with the increasing Experimental design age of the tiger groupers. However, there was no signifi- At the start of the experiment, the remaining fifteen ju- cant (p > 0.05) increase in the numbers of villi in the venile tiger groupers from each group were euthanized mid intestine between 90 and 120 days old tiger grouper. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 3 of 13 http://www.springerplus.com/content/2/1/611 g d e e a f f Groups Figure 1 Number of villi in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). Similar observation was noted for the posterior region of μm for tiger groupers of 30, 60, 90 and 120 days old, the 60 and 90 days old tiger grouper (Figure 1). respectively. The average length of villi at the mid intes- tine was 89.094 ± 10.205, 189.96 ± 34.111, 215.03 ± Length of villi 34.326 and 365.99 ± 39.110 μm at 30, 60, 90 and Measurements of the length of villi revealed significant 120 days old, respectively. The average number of villi (p < 0.05) differences between the intestinal regions of at the posterior intestine was 60.605 ± 20.576, 143.03 ± all group tiger groupers (Figure 3). The average length 35.463, 185.31 ± 59.198 and 265.44 ± 46.064 μm, re- of villi at the anterior intestine was 117.08 ± 23.509, spectively. For all groups, the length was significantly 234.75 ± 42.025, 356.21 ± 59.268 and 400.55 ± 31.133 (p < 0.05) highest in the anterior region, followed by mid Figure 2 Cross-section of anterior intestine of 90 days old tiger grouper. High numbers of villi were observed (arrows) and it is important to make the absorption process very effective (H&E x10). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 4 of 13 http://www.springerplus.com/content/2/1/611 g g e e Groups Figure 3 Length of villi in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). region and least in the posterior intestine. Nevertheless, anterior intestine (Figures 5 and 6). The average gap be- the length showed gradual increase with increasing age tween villi of the anterior intestine was 30.143 ± 4.0889, of the tiger groupers (Figures 3 and 4). 16.756 ± 2.7046, 14.665 ± 2.3584 and 13.606 ± 2.292 μm for tiger grouper at 30, 60, 90 and 120 days old, respect- Gap between villi ively. The average gap between villi of the mid intestine Widest villus gap was observed in the posterior intestine was 41.690 ± 7.2816, 27.275 ± 5.5747, 21.957 ± 4.9491 followed by the mid and the smallest gap was in the and 21.888 ± 2.9411 μm at 30, 60, 90 and 120 days old, Figure 4 Cross-section of posterior intestine of 90 days old tiger grouper. The villi in posterior region is shortest compared to in mid and anterior intestine (arrows) (H&E x20). Length Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 5 of 13 http://www.springerplus.com/content/2/1/611 a e ae h h d g Groups Figure 5 Cross-section of mid intestine of 120 days old tiger grouper showing intermediately tall villi with lamina propria (arrows) that is thicker than anterior intestine but thinner than the posterior intestine (H & E ×40). respectively. The average gap between villi of the posterior tiger grouper, except between 30 and 60 days old tiger intestine was 66.024 ± 13.096, 38.078 ± 5.7424, 29.258 ± groupers (Figure 6). 6.1213 and 30.604 ± 4.9327 μm, respectively. Villus gap in the posterior region was significantly Thickness of lamina propria (p < 0.05) wider than the mid and anterior regions. In all Lamina propria in the anterior region was found to be regions of the intestine, the gap was gradually and sig- significantly (p < 0.05) thin, followed by the mid region nificantly (p < 0.05) decreasing with increasing age of and significantly (p < 0.05) thick at the posterior region. Figure 6 Cross-section of anterior intestine of 30 days old tiger grouper. The size of gap between villi in 30 days old tiger grouper is wide (arrows) and reduces when age is increase (H&E x20). Length of gap Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 6 of 13 http://www.springerplus.com/content/2/1/611 This was observed in all groups of tiger groupers (Figure 7). old, respectively. The average number of lymphoid cells in The average thickness of lamina propria at the anterior the mid intestine was 22.600 ± 3.8508, 65.933 ± 10.807, intestine was 14.854 ± 3.7412, 24.197 ± 3.9502, 25.889 ± 107.53 ± 17.635 and 192.67 ± 35.826 μm at 30 days, 4.2674 and 25.792 ± 2.8590 μm for tiger groupers at 30, 60, 60 days, 90 days and 120 days old, respectively. The aver- 90 and 120 days old, respectively. The average thickness of age number of lymphoid cells in the posterior intestine lamina propria at the mid intestine was 22.435 ± 3.4695, was 32.867 ± 8.1141, 97.600 ± 28.362, 145.33 ± 26.300 and 31.094 ± 5.0122, 33.002 ± 5.7754 and 33.514 ± 3.8933 μmat 268.07 ± 52.138 μm, respectively. 30, 60, 90 and 120 days old, respectively (Figure 8). The The numbers of lymphoid cells in lamina propria average thickness of lamina propria at the posterior intes- were significantly (p < 0.05) highest in the posterior re- tine was 27.947 ± 6.9433, 34.598 ± 7.6659, 38.509 ± 7.6971 gion, followed by the mid region and least in the an- and 39.415 ± 7.1762 μm, respectively. terior region. The number of lymphoid cells in the The thickness of lamina propria in all regions of intes- intestine of 30 days old tiger groupers was significantly tine was observed to gradually increased with age. How- (p < 0.05) lower than other studied ages. Similarly, the ever, significant (p < 0.05) difference in the thickness of average number of lymphoid cells in the intestine of lamina propria was only observed between 30 days old 60 days old was significantly (p < 0.05) lower than the and other groups of 60, 90 and 120 days old tiger grouper 90 days old, which was significantly (p < 0.05) lower in all regions of intestine. The differences in thickness of than the 120 days old (Figure 9). The number of lamina propria among 60, 90 and 120 days old tiger lymphoid cells in all intestinal regions was observed to grouper was found insignificant (p > 0.05) in all regions of gradually increase with age. the intestine. Number of goblet cells Numbers of lymphoid cells At the early age, goblet cell counts showed no signifi- The numbers of lymphoid cells in the lamina propria cant (p > 0.05) differences in all regions of the intestine were found to differ between the different intestinal re- as observed in the 30 and 60 days old tiger grouper gions. Significantly (p < 0.05) highest average number of (Figures 11 and 12). The 30 days old groupers had an lymphoid cells was found in the posterior intestine average number of 11.467 ± 3.2704, 13.000 ± 2.4785 and followed by the mid and the anterior intestines of all age 13.067 ± 2.5204 cells in the anterior, mid and posterior groups (Figures 9 and 10). The average number of lymph- intestines, respectively compared to 13.200 ± 2.9568, oid cells in the anterior intestine was 18.867 ± 6.1975, 14.600 ± 2.9228 and 14.133 ± 2.3258 cells in the anterior, 52.867 ± 10.218, 83.400 ± 14.657 and 150.40 ± 25.972 μm mid and posterior intestines of 60 days old groupers, for tiger groupers at 30 days, 60 days, 90 days and 120 days respectively. d d cd cd b b Groups Figure 7 Cross-section of posterior intestine of 120 days old tiger grouper showing significantly (p < 0.05) high concentration of lymphoid cells (arrows) in the lamina propria (H & E ×40). Thickness Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 7 of 13 http://www.springerplus.com/content/2/1/611 Figure 8 Cross-section of mid intestine of 120 days old tiger grouper. The thickness of lamina propria in mid intestine (arrows) are thinner than posterior but thicker than anterior (H&E x40). However, the number of goblet cells started to sig- 116.07 ± 14.733 cells in the anterior, 141.00 ± 22.548 nificantly (p < 0.05) increased at the age of 90 days cells in the mid and 112.00 ± 29.653 cells in the pos- old with 54.200 ± 6.2929 cells in the anterior intestine, terior intestines. The number of goblet cells in all re- 77.733 ± 10.117 cells in the mid and 80.600 ± 9.2257 gions of the 90-day old tiger grouper was significantly cells in the posterior intestines. The incremental pat- (p < 0.05) less than the 120-day old tiger groupers tern continued in the 120-day old tiger groupers with (Figure 11). hh f f ab Groups Figure 9 Number of goblet cells in lamina propria of three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 8 of 13 http://www.springerplus.com/content/2/1/611 Figure 10 Cross-section of posterior intestine of 120 days old tiger grouper. High concentration of lymphoid cells found scattered in lamina propria of posterior intestine (arrows) (H&E x40). Thickness of intestinal muscle 90 days old, muscle thickness at the anterior regions The growth of intestinal muscles was found to be slow in (32.717 ± 9.5340 μm and 61.287 ± 18.161 μm, respectively) the first three months before drastically increased at the was significantly (p < 0.05) thicker than the mid regions age of 120 days old (Figure 13). At 30 days, the thickness (27.053 ± 7.6956 μm and 37.535 ± 6.8394 μm, respectively), of muscular layer of the anterior intestine was 18.242 ± but not significant (p > 0.05) than the posterior regions 5.2238 μm, which was significantly (p < 0.05) thicker than (35.241 ± 11.660 μm and 62.425 ± 20.315 μm, respectively). the mid (12.387 ± 3.7875 μm) and posterior (12.637 ± However, observation in 120 days old tiger groupers re- 3.3883 μm) regions (Figure 14). At the ages of 60 and vealed the intestinal muscle to be thickest at the anterior d d cc aa a a a a Groups Figure 11 Number of goblet cells in lamina propria of three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1M), 60 days (2M), 90 days (3M) and 120 days (4M). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 9 of 13 http://www.springerplus.com/content/2/1/611 Figure 12 Cross-section of anterior intestine of 90 days old tiger grouper. Goblet cells (arrows) found in every regions of intestine (H&E x40). region (271.02 ± 57.174 μm), followed by the posterior re- Correlations gion (196.35 ± 23.115 μm) and thinnest in the mid region The number of villi (0.9097), the length of villi (0.9246 μm), (123.72 ± 42.411 μm). the number of lymphoid cells (0.9343), the thickness of Generally, the thickness of intestinal muscle gradually lamina propria (0.6621 μm), the number of goblet cells increased with age. The thickness of 30 days old tiger (0.9178) and the thickness of intestinal muscle (0.8262 μm) grouper was significantly (p < 0.050 less than the 60 days of the anterior intestine showed direct proportionate to the old, which was significantly (p < 0.05) less than the age of tiger grouper and between each other. Only the gap 90 days old and significantly (p < 0.05) less than the between villi (−0.7994 μm) was inversely proportional to 120 days old tiger groupers. the age and other parameters (Additional file 1: Table S1). e e cd c c ab b Groups Figure 13 Thickness of muscle in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1M), 60 days (2M), 90 days (3M) and 120 days (4M). Thickness Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 10 of 13 http://www.springerplus.com/content/2/1/611 Figure 14 Cross-section of anterior intestine of 90 days old tiger grouper. The thickest muscle (arrows) found in anterior region of intestine (H&E x10). Similar correlations were observed in the mid (Additional have a thin wall, about one-cell thick known as enterocyte file 2: Table S2) and posterior (Additional file 3: Table S3) that enables a shorter diffusion path (Ferraris et al., 1990; intestines. Oxley et al., 2007). They also have a large surface area due to their ‘loops-like’ shape, for more efficient absorption of Discussion nutrients into the blood stream (Bakke et al., 2011). The Intestine is a tubular organ where feed from stomach complete shape and function of villi could be observed in passes through to start an alkaline digestion before the nu- as early as 30 days old tiger grouper for absorption of trient absorption (Canan et al., 2012). Unlike mammals, smaller and solubilized nutrients. there are no distinct separation between small and large in- The numbers of villi in the anterior intestine were sig- testines of teleost fish (Albrecht et al., 2001). In other word, nificantly high since it is the place where mechanically the intestine is just an undifferentiated tube. Furthermore, digested feeds or chyme from the stomach started to be the intestine of teleost did not reveal obvious crypts of Lie- absorbed, just like the first filter that filtrate maximum berkuhn that normally found in mammals. However, Wal- amount of digested feed particles from the stomach. lace et al. (2005) divided the intestine into three regions Therefore, the maximum numbers of villi are needed to namely the anterior, mid and posterior regions. do the maximum absorption job (Bakke et al., 2011). The intestinal epithelium is a single-cell layer from the Similarly, the lengths of villi in anterior intestine were type of absorptive columnar cells or enterocytes, which significantly taller. This was to provide more surface area play a role in protecting the fish against harmful agents for absorption of nutrient-rich feed particles more effi- in lumen. It also functions as a site for nutrient, water ciently (Nordrum et al., 2000; Bakke et al., 2010). This and ion uptake (Sundell et al., 2003). In fish, the length was in agreement with the conclusion that absorption of of intestine varies and depends on the diet, but basically nutrients such as protein, carbohydrate and lipid oc- between 0.4 and 38 times longer than the body length. curred at a faster rate in proximal or anterior regions of The amount of plant materials in diet is the major deter- intestine (Collie, 1985; Buddington and Diamond 1987; mination factor for intestinal length. Usually, herbivor- Dabrowski, 1990; Bakke-McKellep et al., 2000; Jutfelt ous fish have longer intestine compared to carnivorous et al., 2007). fish (Clements and Raubenheimer, 2006). The remnants of feed particles that are not absorbed This study elaborates the evolution of intestinal tissues in the anterior intestine then migrate into mid intestine where absorption process continues to occur. Since the and cells of tiger grouper from the age of 30 days until 120 days, which included the villi, lamina propria, lymph- amounts of feed particles that migrate toward mid intes- oid cells, goblet cells and muscle tissues. Villi are small, tine are lesser, the number and length of villi were sig- nificantly reduced. Finally, the remaining unabsorbed finger-like projections that protrude from the epithelial lining of the intestinal wall. Villi are specialized tissues, feed particles and wastes migrate into posterior intestine, created to do the absorption in the small intestine as they waiting to be removed from the body through anus that Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 11 of 13 http://www.springerplus.com/content/2/1/611 is located at the end of posterior intestine. Thus, the The function of the villous gap is to provide spaces for function of posterior intestine or hindgut is more to im- food particles to be absorbed by the villi. Therefore, the munity than to absorption process (Ezeasor and Stokoe gap in 30 days old tiger grouper was significantly larger 1981; Buddington and Diamond, 1987). Since the feed due to the feeding behavior where young tiger grouper demand and consumption increase with age of fish, the usually consume less food. number and length of villi keep increasing with age of Goblet cells are glandular simple columnar epithelial the tiger grouper. cells and the major mucous cell type in the intestine of The lamina propria is a vascularized connective tissue fishes (Wilson and Castro, 2011). The main function of containing nerves and leukocytes, which lies beneath the goblet cells is to secrete mucin that dissolves in water to epithelium and together with the epithelium constitutes form mucus, a clear, colorless and slimy substance that the mucosa (Wilson and Castro, 2011). The thickness of creates a layer to coat the wall of intestine (Kim and lamina propria in anterior intestine was significantly less Samuel, 2010). Besides function as a lubricant for smooth than the mid intestine but became significantly thicker movement of feed particles, mucus also provides innate in the posterior intestine. Therefore, the thickness of host defense by acting as a first line of immunity against in- lamina propria was inversely proportional with the num- vasion of harmful pathogen (Kim and Samuel, 2010). Intes- ber of villi. The rapid growth and development of the tinal mucus secreted in fish contains antibody (Grabowski lamina propria occurred between 30-day and 60-day old et al., 2004; Firdaus-Nawi et al., 2012), lysozyme (Lie et al., groupers when significant increase in the thickness of 1989), glycoprotein (Shephard, 1994), complement compo- lamina propria was observed. nents, lectins and some antimicrobial agents (Ingram, This study also revealed that the thickness of lamina propria 1980; Alexander and Ingram, 1992). In this study, goblet was related with the concentration of lymphoid cells and the cells were found scattered among the epithelial lining in all region’s specific function. High concentrations of lymphoid three regions of the intestine, but showed no significant cells were observed in the posterior intestine, and support the difference in numbers until 90 days old. Between 90 and conclusion that posterior intestine plays a major role in im- 120 days old, the numbers of goblet cells in mid intestine munity (Buddington and Diamond, 1987). Similarly, Ezeasor were dramatically increased, suggesting the important non- and Stokoe (1981) found high phagocytic activities in the pos- specific or innate immunity role played by the region. terior intestine compared to another regions while a study in Therefore, the innate immune system in the mid intestine red tilapia revealed accumulation of lymphoid cells in the detects and prevents the presence of invasive pathogen lamina propria of posterior intestine following oral vaccin- prior to stimulation of the specific immune system in the ation with adjuvanted feed-base vaccine against streptococco- posterior intestine. sis (Firdaus-Nawi et al., 2012). Due to the immunity functions Teleost intestines are lack of muscularis mucosa that but less nutrient absorptive capacity (Buddington and Dia- divides lamina propria from sub-mucosa (Wallace et al., mond, 1987), posterior intestine requires large numbers of 2005). The development of intestinal muscle can be lymphoid cells and the lamina propria should be thick to pro- attributed to the feeding regime. From birth to the age vide enough space for the lymphoid cells. of 40 days old, tiger grouper were fed with live feed since The thickness of lamina propria and number lymphoid their digestion system; particularly due to intestinal cells were significantly reduced in the mid intestine. Al- muscle which less developed. This makes oral vaccin- though the function of mid intestine is not well under- ation via live feed possible for juvenile groupers At the stood, it was suggested that mid intestine moderately age of 40 days old and above, they were fed with larvae play both absorption and immunity roles. Mid intestine pellet where the intestinal muscle became thicker as ob- completes the absorption process that was previously done served in the 60 and 90 days old tiger groupers. Large- by anterior intestine and starts the immune response prior sized pellet was introduced to the tiger grouper at the age of 100 days, thus the thickest muscular layer was ob- to the posterior intestine. The presence of lymphoid and goblet cells in intestines of 30-day old tiger grouper indi- served in tiger grouper at age 120 days old. cated that intestinal immunity was present at that particular age. This is in agreement with previous study by Lin et al. Conclusion (2007) in 19-day old groupers where oral immunization From the study, we have found the intestinal immunity in with inactivated nervous necrosis virus (NNV) provided tiger grouper is existed as early as thirty days old age, and good protection after challenge with live NNV. every region of intestine have different roles either in food Empty space between two villi is called villous gap that processing or immunity. Since the intestinal immunity is appears to be influenced by the number of villi. Size of existed at the age of thirty days old, vaccination could be the gap in region that was packed with villi, such as the conducted at that particular age. However, vaccination at anterior intestine, was narrower than the size in mid and 60-day old is expected to stimulate stronger immune re- posterior intestines where the number of villi was less. sponse since the immune cells were well establsihed. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 12 of 13 http://www.springerplus.com/content/2/1/611 Additional files challenged with infectious hematopoietic necrosis virus: identification of antiviral activity. Dis Aquat Organ 27:161–172 Cain KD, Jones DR, Raison RL (2000) Characterization of mucosal and systemic Additional file 1: Table S1. Correlation (Pearson) value between each immune responses in rainbow trout (Oncorhynchus mykiss) using surface studied parameters in anterior intestine, age of tiger grouper (AGE), gap plasmon resonance. Fish Shellfish Immunol 10:651–666 between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Canan B, Do Nascimento WS, Da Silva NB, Chellappa S (2012) Morphohistology of number of goblet cells (NGC), number of villi (NOV), number of lymphoid the digestive tract of the damsel fish Stegastes fuscus (Osteichthyes: cells (NLC) and thickness of muscle (TOM). Pomacentridae). The Scientific World Journal Article ID 787316:9, Additional file 2: Table S2. Correlation (Pearson) value between each doi:10.1100/2012/787316 studied parameters in mid intestine, age of tiger grouper (AGE), gap Clements KD, Raubenheimer D (2006) Feeding and Nutrition. In: Evans DH, between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Claiborne JB (eds) The physiology of fishes. Taylor and Francis Group, Boca number of goblet cells (NGC), number of villi (NOV), number of lymphoid Raton, FL, USA cells (NLC) and thickness of muscle (TOM). Cole AM, Weis P, Diamond G (1997) Isolation and characterization of pleurocidin, Additional file 3: Table S3. Correlation (Pearson) value between each an antimicrobial peptide in the skin secretions of winter flounder. J Biol studied parameters in posterior intestine, age of tiger grouper (AGE), gap Chem 272:12008–12013 between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Collie NL (1985) Intestinal nutrient transport in coho salmon (Oncorhynchus number of goblet cells (NGC), number of villi (NOV), number of lymphoid kisutch) and the effects of development, starvation, and seawater adaptation. cells (NLC) and thickness of muscle (TOM). J Comp Physiol B 156:163–174 Concha MI, Molina S, Oyarzun C, Villanueva J, Amthauer R (2003) Local expression of apolipoprotein A-I gene and a possible role for HDL in primary Competing interests defence in the carp skin. Fish Shellfish Immunol 14:259–73 The authors declare that they have no competing interests. Dabrowski K (1990) Absorption of ascorbic acid andascorbic sulfate and ascorbate metabolism in stomachless fish, Common carp. J Comp Physiol B Authors’ contribution 160:549–561 MFN has carried out the research work and analyzed the date, MZS outlined Dalmo RA, Ingebrightsen K, Bogwald J (1997) Non-specific defense mechanisms the research design and supervised the experimental trial, MZAB and MEAW in fish, with particular reference to the reticuloendothelial system (RES). J Fish helped revised the project and read the manuscript. All authors read and Dis 20:241–273 approved the final manuscript. De Silva SS (1998) Tropical Mariculture: Current Status and Prospects. Trop Mariculture 1:1–16 Ellis AE (2001) The immunology of teleosts. In: Roberts RJ (ed) Fish Pathology. WB Acknowledgement Saunders, London, pp 133–150 The authors wish to thank the Marine Aquaculture Breeding Technology Ezeasor DN, Stokoe WM (1981) Light and electron microscopic studies on the Centre, Tanjung Demong, Terengganu, Malaysia for fish supply and facilities absorptive cells of the intestine ceca and rectum of the adult rainbow trout, for conducting this study. We thank Ahmad Baihaqi Othman and Dr. Siti Salmo gairdneri. J Fish Biol 18:527–544 Zahrah Abdullah for their technical assistance and advice. Federal Agricultural Marketing Authority of Malaysia (2013) National Agro-food Author details Policy 2010–2020., Accessed on October 26, 2013 at http://www.fama.gov. Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor my/en/dasar-agromakanan-negara-2011-2020-dan-#.UpSxQo0Vg3Y 43400, Malaysia. Marine Finfish Production and Research Centre, FRI Tanjung Fernandes JMO, Kemp GD, Smith VJ (2004) Two novel muramidases from skin Demong, Besut, Terengganu 22200, Malaysia. Institute of Marine mucosa of rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu, B 138:53–64 Terengganu 21030, Malaysia. Ferraris RP, Yasharpour S, Lloyd KC, Mirzayan R, Diamond JM (1990) Luminal glucose concentrations in the gut under normal conditions. Am J Physiol Received: 3 October 2013 Accepted: 7 November 2013 259:G822–G837 Published: 15 November 2013 Firdaus-Nawi M, Sabri MY, Hanan Y, Siti-Zahrah A, Zamri-Saad S (2012) Efficacy of feed-based adjuvant vaccine against Streptococcus agalactiae in Oreo- chromis spp. in Malaysia. Aquacult Res, In Press (Early view) doi:10.1111/ References j.1365-2109.2012.03207.x Afero F, Miao S, Perez AA (2009) Economic analysis of tiger grouper Epinephelus Fletcher TC, Grant PT (1968) Glycoproteins in the external mucous secretions of fuscoguttatus and humpback grouper Cromileptes altivelis commercial cage the plaice, Pleuronectes platessa and some other fishes. Biochemistry 106:12 culture in Indonesia. Aquac Int, doi:10.1007/s10499- 009-9295-x Food and Agriculture Organization (FAO) (2013) FAO Statistical Yearbook-World Albrecht MP, Ferreira MFN, Caramaschi EP (2001) Anatomical features and Food And Agriculture 2013. FAO Publishing, Food and Agriculture histology of the digestive tract of two related neotropical omnivorous fishes Organization of the United Nations, Rome, Italy, ISSN: 2225-7373 (Characiformes; Anostomidae). 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Thai Fisheries Gazette, Thai National AGRIS Centre (Thailand), Main Library, Kasertsart University, PO Box 1084, Kasertsart Chatuchak, Bangkok 10903, Thailand doi:10.1186/2193-1801-2-611 Cite this article as: Firdaus-Nawi et al.: Histological assessments of intestinal immuno-morphology of tiger grouper juvenile, Epinephelus fuscoguttatus. SpringerPlus 2013 2:611. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png SpringerPlus Springer Journals

Histological assessments of intestinal immuno-morphology of tiger grouper juvenile, Epinephelus fuscoguttatus

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Copyright © 2013 by Firdaus-Nawi et al.; licensee Springer.
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10.1186/2193-1801-2-611
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Abstract

Histological assessments on the intestinal morphology and immunity of tiger grouper juveniles, Epinephelus fuscoguttatus help in determining the earliest age to start an oral vaccination. This study describes the morphological development of the intestinal immunity of tiger grouper of various ages. Clinically healthy tiger groupers were selected and divided into 4 groups of 20 fish per group. Groups 1, 2, 3 and 4 consisted of juveniles of 30, 60, 90 and 120 days old, respectively. The whole intestine was collected and divided into three regions, the anterior, mid and posterior intestine and fixed in 10% buffered formalin before slides were prepared for microscopic examinations. It was found that the histological structures of the anterior intestine were for absorption of nutrient from digested food particles. The significantly (p < 0.05) higher number and length of the intestinal villi and smaller gap between villi were observed in the anterior intestine, which were structures for absorption. Structures of the posterior intestine were for immunity especially the adaptive immunity with included significantly (p < 0.05) higher numbers of the lymphoid and goblet cells, and significantly (p < 0.05) thicker lamina propria, which were structures for immunity. The mid intestine was the transition structure that involved in both absorption and innate immunity. The results also revealed that leukocytes existed in the lamina propria of 30-days old tiger groupers, an indication that the immune system was present at that particular age. Keywords: Tiger grouper; Intestine histological; Assessment; Immune system Introduction artisanal fisheries, which groupers are the major compo- Grouper is a high value cultured marine fish especially in nent. According to Food and Agriculture Organization the Southeast Asia countries including Malaysia, Indonesia, (FAO) (2013), world aqu of (2880saculture production of Thailand and the Philippines (Liao et al., 2001; Yashiro, groupers was around 6000–7000 tones per annum, valued 2008). The major cultured species are giant grouper, Epine- at about USD60 million and the bulk of this production phelus lanceolatus (Yashiro, 2008), tiger grouper, Epinephe- came from wild seed stock due to lack of reared seed that lus fuscoguttatus (Sugama et al., 2008), malabar grouper, suffered disease problems. Epinephelus malabaricus (Yashiro, 2008), orange spotted In Malaysia, tiger grouper, Epinephelus fuscoguttatus, grouper, Epinephelus coioides (Toledo et al. 1996), hump- also known as brown marbled grouper, is a major cul- back grouper, Cromileptes altivelis (Marte, 2003), leopard tured marine species. It is considered a popular and coralgrouper, Plectropomus leopardus (De Silva, 1998) and high valued aquaculture species and fast growing (Afero hybrid grouper, (Epinephelus lanceolatus X Epinephelus et al., 2009). Data from Federal Agricultural Marketing fuscoguttatus). Heemstra and Randall (1993) estimated that Authority of Malaysia (2013) (FAMA) indicated that 90% of the world’s harvest on marine food is derived from grouper species are the major export fish for the year 2010 (MAHA, 2013). Fish digestive system is slightly differ from mammal. The * Correspondence: zamri@vet.upm.edu.my Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor digestive process has started in the first region that in- 43400, Malaysia cludes the mouth, oral cavity, and pharynx. Then, after Full list of author information is available at the end of the article © 2013 Firdaus-Nawi et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 2 of 13 http://www.springerplus.com/content/2/1/611 food is swallowed it will enter the alimentary canal proper by an overdose of Ethyl 3-aminobenzoate methanesulfo- and proceeds via the esophagus to the stomach followed nate (Sigma Aldrich, USA). Post-mortem examination by the intestines (Buddington and Kuz’mina, 2000). The was performed immediately; the entire intestine was re- main function of fish intestine is to complete the digestive moved and was cut into the anterior, mid and posterior process, which started in the stomach and also to absorb portions (Wilson and Castro, 2011) before the intestinal the nutrients from food (Wilson and Castro, 2011). Other samples were fixed into 10% buffered formaldehyde for than the digestive function, gut of fish also acts as the first- at least 24 h. The Animal Care and Use Committee of line barrier against infection. Thus, the mucosal layer of University Putra Malaysia approved the study protocol. the gut creates physical, chemical and cellular protections against pathogen invasions (Ellis, 2001). The goblet cells Sample preparation and histological analysis and the glandular simple columnar epithelial cells se- Following 24 h fixation, the samples were prepared for crete mucus, which contains immunological substances histological examination according to Firdaus-Nawi et al. such as glycoprotein (Fletcher and Grant, 1968), cyto- (2012). The slides were examined under a light micros- kines (Lindenstrøm et al. 2003), peptides (Cole et al., copy using Nikon NIS-Element D 3.2 Image Analyzer 1997), lysozyme (Fernandes et al., 2004), lipoprotein (Nikon Instruments Inc., USA). For each slide, a total of (Concha et al., 2003), complement (Dalmo et al., 1997), five microscopic fields were examined at 200× magnifica- lectins (Tsutsui et al., 2005), proteases (Aranishi and Mano, tion to determine the number of villi, the length of villi, 2000) and antibodies (Cain et al., 2000). These substances the gap between villi, the thickness of lamina propria, the provide direct or indirect protection against pathogen number of lymphoid cells, the number of goblet cells and (Cain et al., 1996). Furthermore, the intestine of fish con- the thickness of muscular layer. The Nikon NIS-Element tains lymphoid cells that secrete antibodies and involve in D 3.2 Image Analyzer (Nikon Instruments Inc., USA) was phagocytosis. The aim of this study is to describe the histo- used to measure all parameters. logical evaluation on the intestinal immune-morphology of tiger grouper, Epinephelus fuscoguttatus juveniles. Statistical analysis The Statistix 9 (Analytical Software, USA) was employed Materials and methods to analyze the data. The results revealed significance in Fish and rearing conditions all pairwise comparison under one-way ANOVA. Signifi- A total of 80 healthy tiger grouper (Epeniphelus fuscogutta- cant differences were determined at P < 0.05. Pearson tus) juveniles of different ages were used. They were 30, 60, correlation test was used to reveal the correlation be- 90 and 120 days old of approximately 1 g ± 0.3 g, 2.5 g ± tween each studied parameters. 0.3 g, 200 g ± 0.3 g and 300 g ± 0.3 g body weight, and of approximately 2.5 cm ± 0.5 cm, 5 cm ± 0.5 cm, 7.5 cm ± Results 0.5 cm and 10 cm ± 0.5 cm in length, respectively. The se- Number of villi lected juveniles were grouped according to the age with 20 There were significant (p < 0.05) differences in the numbers fish per group and kept separately in 100-L glass tanks. of villi in the three regions of intestine (Figure 1). The aver- Fish of less than 30 days old were too small for handling age number of villi of anterior intestine was 39.200 ± and sampling. All the fish were acclimatized for at least 6.1435, 64.467 ± 11.077, 74.333 ± 11.709 and 95.533 ± 7 days prior to experiment. Light cycle was held constant 4.0860 for tiger groupers at 30, 60, 90 and 120 days old, with 12 hours of lighting per day. Feeding was ad libitum respectively. The average number of villi of mid intestine with a local commercial feed while water was continuously was 22.533 ± 3.6227, 49.933 ± 2.8149, 57.867 ± 6.8334 and aerated. The water temperature, pH, salinity and dissolved 62.000 ± 8.000 μm at 30, 60, 90 and 120 days old, re- oxygen were measured daily using the HQ40d Meter spectively. The average number of villi of posterior in- (Hach Company, Loveland, CO). The ammonia, sulfate testine was 29.067 ± 5.1195, 43.333 ± 4.7006, 44.533 ± and nitrites were determined daily using the DR 2800 Port- 3.3989 and 51.467 ± 4.8236 μm, respectively. able Spectrophotometer (Hach Company, Loveland, CO). The number of villi was highest in the anterior region Prior to sampling, five fish from each age group were sacri- of intestine (Figure 2), followed by the mid region and ficed and swab samples from the organs were collected for least in the posterior region, except for the 30 days old bacterial and parasitic examinations. This was to ensure tiger grouper that showed that the number of villi was that the fish were free from bacterial and parasitic diseases significantly (p < 0.05) higher in the posterior than the (Firdaus-Nawi et al., 2012). mid region. In general, the number of villi showed grad- ual and significant (p < 0.05) increase with the increasing Experimental design age of the tiger groupers. However, there was no signifi- At the start of the experiment, the remaining fifteen ju- cant (p > 0.05) increase in the numbers of villi in the venile tiger groupers from each group were euthanized mid intestine between 90 and 120 days old tiger grouper. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 3 of 13 http://www.springerplus.com/content/2/1/611 g d e e a f f Groups Figure 1 Number of villi in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). Similar observation was noted for the posterior region of μm for tiger groupers of 30, 60, 90 and 120 days old, the 60 and 90 days old tiger grouper (Figure 1). respectively. The average length of villi at the mid intes- tine was 89.094 ± 10.205, 189.96 ± 34.111, 215.03 ± Length of villi 34.326 and 365.99 ± 39.110 μm at 30, 60, 90 and Measurements of the length of villi revealed significant 120 days old, respectively. The average number of villi (p < 0.05) differences between the intestinal regions of at the posterior intestine was 60.605 ± 20.576, 143.03 ± all group tiger groupers (Figure 3). The average length 35.463, 185.31 ± 59.198 and 265.44 ± 46.064 μm, re- of villi at the anterior intestine was 117.08 ± 23.509, spectively. For all groups, the length was significantly 234.75 ± 42.025, 356.21 ± 59.268 and 400.55 ± 31.133 (p < 0.05) highest in the anterior region, followed by mid Figure 2 Cross-section of anterior intestine of 90 days old tiger grouper. High numbers of villi were observed (arrows) and it is important to make the absorption process very effective (H&E x10). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 4 of 13 http://www.springerplus.com/content/2/1/611 g g e e Groups Figure 3 Length of villi in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). region and least in the posterior intestine. Nevertheless, anterior intestine (Figures 5 and 6). The average gap be- the length showed gradual increase with increasing age tween villi of the anterior intestine was 30.143 ± 4.0889, of the tiger groupers (Figures 3 and 4). 16.756 ± 2.7046, 14.665 ± 2.3584 and 13.606 ± 2.292 μm for tiger grouper at 30, 60, 90 and 120 days old, respect- Gap between villi ively. The average gap between villi of the mid intestine Widest villus gap was observed in the posterior intestine was 41.690 ± 7.2816, 27.275 ± 5.5747, 21.957 ± 4.9491 followed by the mid and the smallest gap was in the and 21.888 ± 2.9411 μm at 30, 60, 90 and 120 days old, Figure 4 Cross-section of posterior intestine of 90 days old tiger grouper. The villi in posterior region is shortest compared to in mid and anterior intestine (arrows) (H&E x20). Length Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 5 of 13 http://www.springerplus.com/content/2/1/611 a e ae h h d g Groups Figure 5 Cross-section of mid intestine of 120 days old tiger grouper showing intermediately tall villi with lamina propria (arrows) that is thicker than anterior intestine but thinner than the posterior intestine (H & E ×40). respectively. The average gap between villi of the posterior tiger grouper, except between 30 and 60 days old tiger intestine was 66.024 ± 13.096, 38.078 ± 5.7424, 29.258 ± groupers (Figure 6). 6.1213 and 30.604 ± 4.9327 μm, respectively. Villus gap in the posterior region was significantly Thickness of lamina propria (p < 0.05) wider than the mid and anterior regions. In all Lamina propria in the anterior region was found to be regions of the intestine, the gap was gradually and sig- significantly (p < 0.05) thin, followed by the mid region nificantly (p < 0.05) decreasing with increasing age of and significantly (p < 0.05) thick at the posterior region. Figure 6 Cross-section of anterior intestine of 30 days old tiger grouper. The size of gap between villi in 30 days old tiger grouper is wide (arrows) and reduces when age is increase (H&E x20). Length of gap Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 6 of 13 http://www.springerplus.com/content/2/1/611 This was observed in all groups of tiger groupers (Figure 7). old, respectively. The average number of lymphoid cells in The average thickness of lamina propria at the anterior the mid intestine was 22.600 ± 3.8508, 65.933 ± 10.807, intestine was 14.854 ± 3.7412, 24.197 ± 3.9502, 25.889 ± 107.53 ± 17.635 and 192.67 ± 35.826 μm at 30 days, 4.2674 and 25.792 ± 2.8590 μm for tiger groupers at 30, 60, 60 days, 90 days and 120 days old, respectively. The aver- 90 and 120 days old, respectively. The average thickness of age number of lymphoid cells in the posterior intestine lamina propria at the mid intestine was 22.435 ± 3.4695, was 32.867 ± 8.1141, 97.600 ± 28.362, 145.33 ± 26.300 and 31.094 ± 5.0122, 33.002 ± 5.7754 and 33.514 ± 3.8933 μmat 268.07 ± 52.138 μm, respectively. 30, 60, 90 and 120 days old, respectively (Figure 8). The The numbers of lymphoid cells in lamina propria average thickness of lamina propria at the posterior intes- were significantly (p < 0.05) highest in the posterior re- tine was 27.947 ± 6.9433, 34.598 ± 7.6659, 38.509 ± 7.6971 gion, followed by the mid region and least in the an- and 39.415 ± 7.1762 μm, respectively. terior region. The number of lymphoid cells in the The thickness of lamina propria in all regions of intes- intestine of 30 days old tiger groupers was significantly tine was observed to gradually increased with age. How- (p < 0.05) lower than other studied ages. Similarly, the ever, significant (p < 0.05) difference in the thickness of average number of lymphoid cells in the intestine of lamina propria was only observed between 30 days old 60 days old was significantly (p < 0.05) lower than the and other groups of 60, 90 and 120 days old tiger grouper 90 days old, which was significantly (p < 0.05) lower in all regions of intestine. The differences in thickness of than the 120 days old (Figure 9). The number of lamina propria among 60, 90 and 120 days old tiger lymphoid cells in all intestinal regions was observed to grouper was found insignificant (p > 0.05) in all regions of gradually increase with age. the intestine. Number of goblet cells Numbers of lymphoid cells At the early age, goblet cell counts showed no signifi- The numbers of lymphoid cells in the lamina propria cant (p > 0.05) differences in all regions of the intestine were found to differ between the different intestinal re- as observed in the 30 and 60 days old tiger grouper gions. Significantly (p < 0.05) highest average number of (Figures 11 and 12). The 30 days old groupers had an lymphoid cells was found in the posterior intestine average number of 11.467 ± 3.2704, 13.000 ± 2.4785 and followed by the mid and the anterior intestines of all age 13.067 ± 2.5204 cells in the anterior, mid and posterior groups (Figures 9 and 10). The average number of lymph- intestines, respectively compared to 13.200 ± 2.9568, oid cells in the anterior intestine was 18.867 ± 6.1975, 14.600 ± 2.9228 and 14.133 ± 2.3258 cells in the anterior, 52.867 ± 10.218, 83.400 ± 14.657 and 150.40 ± 25.972 μm mid and posterior intestines of 60 days old groupers, for tiger groupers at 30 days, 60 days, 90 days and 120 days respectively. d d cd cd b b Groups Figure 7 Cross-section of posterior intestine of 120 days old tiger grouper showing significantly (p < 0.05) high concentration of lymphoid cells (arrows) in the lamina propria (H & E ×40). Thickness Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 7 of 13 http://www.springerplus.com/content/2/1/611 Figure 8 Cross-section of mid intestine of 120 days old tiger grouper. The thickness of lamina propria in mid intestine (arrows) are thinner than posterior but thicker than anterior (H&E x40). However, the number of goblet cells started to sig- 116.07 ± 14.733 cells in the anterior, 141.00 ± 22.548 nificantly (p < 0.05) increased at the age of 90 days cells in the mid and 112.00 ± 29.653 cells in the pos- old with 54.200 ± 6.2929 cells in the anterior intestine, terior intestines. The number of goblet cells in all re- 77.733 ± 10.117 cells in the mid and 80.600 ± 9.2257 gions of the 90-day old tiger grouper was significantly cells in the posterior intestines. The incremental pat- (p < 0.05) less than the 120-day old tiger groupers tern continued in the 120-day old tiger groupers with (Figure 11). hh f f ab Groups Figure 9 Number of goblet cells in lamina propria of three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1 M), 60 days (2 M), 90 days (3 M) and 120 days (4 M). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 8 of 13 http://www.springerplus.com/content/2/1/611 Figure 10 Cross-section of posterior intestine of 120 days old tiger grouper. High concentration of lymphoid cells found scattered in lamina propria of posterior intestine (arrows) (H&E x40). Thickness of intestinal muscle 90 days old, muscle thickness at the anterior regions The growth of intestinal muscles was found to be slow in (32.717 ± 9.5340 μm and 61.287 ± 18.161 μm, respectively) the first three months before drastically increased at the was significantly (p < 0.05) thicker than the mid regions age of 120 days old (Figure 13). At 30 days, the thickness (27.053 ± 7.6956 μm and 37.535 ± 6.8394 μm, respectively), of muscular layer of the anterior intestine was 18.242 ± but not significant (p > 0.05) than the posterior regions 5.2238 μm, which was significantly (p < 0.05) thicker than (35.241 ± 11.660 μm and 62.425 ± 20.315 μm, respectively). the mid (12.387 ± 3.7875 μm) and posterior (12.637 ± However, observation in 120 days old tiger groupers re- 3.3883 μm) regions (Figure 14). At the ages of 60 and vealed the intestinal muscle to be thickest at the anterior d d cc aa a a a a Groups Figure 11 Number of goblet cells in lamina propria of three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1M), 60 days (2M), 90 days (3M) and 120 days (4M). Number Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 9 of 13 http://www.springerplus.com/content/2/1/611 Figure 12 Cross-section of anterior intestine of 90 days old tiger grouper. Goblet cells (arrows) found in every regions of intestine (H&E x40). region (271.02 ± 57.174 μm), followed by the posterior re- Correlations gion (196.35 ± 23.115 μm) and thinnest in the mid region The number of villi (0.9097), the length of villi (0.9246 μm), (123.72 ± 42.411 μm). the number of lymphoid cells (0.9343), the thickness of Generally, the thickness of intestinal muscle gradually lamina propria (0.6621 μm), the number of goblet cells increased with age. The thickness of 30 days old tiger (0.9178) and the thickness of intestinal muscle (0.8262 μm) grouper was significantly (p < 0.050 less than the 60 days of the anterior intestine showed direct proportionate to the old, which was significantly (p < 0.05) less than the age of tiger grouper and between each other. Only the gap 90 days old and significantly (p < 0.05) less than the between villi (−0.7994 μm) was inversely proportional to 120 days old tiger groupers. the age and other parameters (Additional file 1: Table S1). e e cd c c ab b Groups Figure 13 Thickness of muscle in three regions of tiger grouper intestine; anterior intestine (AI), mid intestine (IM) and posterior intestine (PI), in four experimental ages; 30 days (1M), 60 days (2M), 90 days (3M) and 120 days (4M). Thickness Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 10 of 13 http://www.springerplus.com/content/2/1/611 Figure 14 Cross-section of anterior intestine of 90 days old tiger grouper. The thickest muscle (arrows) found in anterior region of intestine (H&E x10). Similar correlations were observed in the mid (Additional have a thin wall, about one-cell thick known as enterocyte file 2: Table S2) and posterior (Additional file 3: Table S3) that enables a shorter diffusion path (Ferraris et al., 1990; intestines. Oxley et al., 2007). They also have a large surface area due to their ‘loops-like’ shape, for more efficient absorption of Discussion nutrients into the blood stream (Bakke et al., 2011). The Intestine is a tubular organ where feed from stomach complete shape and function of villi could be observed in passes through to start an alkaline digestion before the nu- as early as 30 days old tiger grouper for absorption of trient absorption (Canan et al., 2012). Unlike mammals, smaller and solubilized nutrients. there are no distinct separation between small and large in- The numbers of villi in the anterior intestine were sig- testines of teleost fish (Albrecht et al., 2001). In other word, nificantly high since it is the place where mechanically the intestine is just an undifferentiated tube. Furthermore, digested feeds or chyme from the stomach started to be the intestine of teleost did not reveal obvious crypts of Lie- absorbed, just like the first filter that filtrate maximum berkuhn that normally found in mammals. However, Wal- amount of digested feed particles from the stomach. lace et al. (2005) divided the intestine into three regions Therefore, the maximum numbers of villi are needed to namely the anterior, mid and posterior regions. do the maximum absorption job (Bakke et al., 2011). The intestinal epithelium is a single-cell layer from the Similarly, the lengths of villi in anterior intestine were type of absorptive columnar cells or enterocytes, which significantly taller. This was to provide more surface area play a role in protecting the fish against harmful agents for absorption of nutrient-rich feed particles more effi- in lumen. It also functions as a site for nutrient, water ciently (Nordrum et al., 2000; Bakke et al., 2010). This and ion uptake (Sundell et al., 2003). In fish, the length was in agreement with the conclusion that absorption of of intestine varies and depends on the diet, but basically nutrients such as protein, carbohydrate and lipid oc- between 0.4 and 38 times longer than the body length. curred at a faster rate in proximal or anterior regions of The amount of plant materials in diet is the major deter- intestine (Collie, 1985; Buddington and Diamond 1987; mination factor for intestinal length. Usually, herbivor- Dabrowski, 1990; Bakke-McKellep et al., 2000; Jutfelt ous fish have longer intestine compared to carnivorous et al., 2007). fish (Clements and Raubenheimer, 2006). The remnants of feed particles that are not absorbed This study elaborates the evolution of intestinal tissues in the anterior intestine then migrate into mid intestine where absorption process continues to occur. Since the and cells of tiger grouper from the age of 30 days until 120 days, which included the villi, lamina propria, lymph- amounts of feed particles that migrate toward mid intes- oid cells, goblet cells and muscle tissues. Villi are small, tine are lesser, the number and length of villi were sig- nificantly reduced. Finally, the remaining unabsorbed finger-like projections that protrude from the epithelial lining of the intestinal wall. Villi are specialized tissues, feed particles and wastes migrate into posterior intestine, created to do the absorption in the small intestine as they waiting to be removed from the body through anus that Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 11 of 13 http://www.springerplus.com/content/2/1/611 is located at the end of posterior intestine. Thus, the The function of the villous gap is to provide spaces for function of posterior intestine or hindgut is more to im- food particles to be absorbed by the villi. Therefore, the munity than to absorption process (Ezeasor and Stokoe gap in 30 days old tiger grouper was significantly larger 1981; Buddington and Diamond, 1987). Since the feed due to the feeding behavior where young tiger grouper demand and consumption increase with age of fish, the usually consume less food. number and length of villi keep increasing with age of Goblet cells are glandular simple columnar epithelial the tiger grouper. cells and the major mucous cell type in the intestine of The lamina propria is a vascularized connective tissue fishes (Wilson and Castro, 2011). The main function of containing nerves and leukocytes, which lies beneath the goblet cells is to secrete mucin that dissolves in water to epithelium and together with the epithelium constitutes form mucus, a clear, colorless and slimy substance that the mucosa (Wilson and Castro, 2011). The thickness of creates a layer to coat the wall of intestine (Kim and lamina propria in anterior intestine was significantly less Samuel, 2010). Besides function as a lubricant for smooth than the mid intestine but became significantly thicker movement of feed particles, mucus also provides innate in the posterior intestine. Therefore, the thickness of host defense by acting as a first line of immunity against in- lamina propria was inversely proportional with the num- vasion of harmful pathogen (Kim and Samuel, 2010). Intes- ber of villi. The rapid growth and development of the tinal mucus secreted in fish contains antibody (Grabowski lamina propria occurred between 30-day and 60-day old et al., 2004; Firdaus-Nawi et al., 2012), lysozyme (Lie et al., groupers when significant increase in the thickness of 1989), glycoprotein (Shephard, 1994), complement compo- lamina propria was observed. nents, lectins and some antimicrobial agents (Ingram, This study also revealed that the thickness of lamina propria 1980; Alexander and Ingram, 1992). In this study, goblet was related with the concentration of lymphoid cells and the cells were found scattered among the epithelial lining in all region’s specific function. High concentrations of lymphoid three regions of the intestine, but showed no significant cells were observed in the posterior intestine, and support the difference in numbers until 90 days old. Between 90 and conclusion that posterior intestine plays a major role in im- 120 days old, the numbers of goblet cells in mid intestine munity (Buddington and Diamond, 1987). Similarly, Ezeasor were dramatically increased, suggesting the important non- and Stokoe (1981) found high phagocytic activities in the pos- specific or innate immunity role played by the region. terior intestine compared to another regions while a study in Therefore, the innate immune system in the mid intestine red tilapia revealed accumulation of lymphoid cells in the detects and prevents the presence of invasive pathogen lamina propria of posterior intestine following oral vaccin- prior to stimulation of the specific immune system in the ation with adjuvanted feed-base vaccine against streptococco- posterior intestine. sis (Firdaus-Nawi et al., 2012). Due to the immunity functions Teleost intestines are lack of muscularis mucosa that but less nutrient absorptive capacity (Buddington and Dia- divides lamina propria from sub-mucosa (Wallace et al., mond, 1987), posterior intestine requires large numbers of 2005). The development of intestinal muscle can be lymphoid cells and the lamina propria should be thick to pro- attributed to the feeding regime. From birth to the age vide enough space for the lymphoid cells. of 40 days old, tiger grouper were fed with live feed since The thickness of lamina propria and number lymphoid their digestion system; particularly due to intestinal cells were significantly reduced in the mid intestine. Al- muscle which less developed. This makes oral vaccin- though the function of mid intestine is not well under- ation via live feed possible for juvenile groupers At the stood, it was suggested that mid intestine moderately age of 40 days old and above, they were fed with larvae play both absorption and immunity roles. Mid intestine pellet where the intestinal muscle became thicker as ob- completes the absorption process that was previously done served in the 60 and 90 days old tiger groupers. Large- by anterior intestine and starts the immune response prior sized pellet was introduced to the tiger grouper at the age of 100 days, thus the thickest muscular layer was ob- to the posterior intestine. The presence of lymphoid and goblet cells in intestines of 30-day old tiger grouper indi- served in tiger grouper at age 120 days old. cated that intestinal immunity was present at that particular age. This is in agreement with previous study by Lin et al. Conclusion (2007) in 19-day old groupers where oral immunization From the study, we have found the intestinal immunity in with inactivated nervous necrosis virus (NNV) provided tiger grouper is existed as early as thirty days old age, and good protection after challenge with live NNV. every region of intestine have different roles either in food Empty space between two villi is called villous gap that processing or immunity. Since the intestinal immunity is appears to be influenced by the number of villi. Size of existed at the age of thirty days old, vaccination could be the gap in region that was packed with villi, such as the conducted at that particular age. However, vaccination at anterior intestine, was narrower than the size in mid and 60-day old is expected to stimulate stronger immune re- posterior intestines where the number of villi was less. sponse since the immune cells were well establsihed. Firdaus-Nawi et al. SpringerPlus 2013, 2:611 Page 12 of 13 http://www.springerplus.com/content/2/1/611 Additional files challenged with infectious hematopoietic necrosis virus: identification of antiviral activity. Dis Aquat Organ 27:161–172 Cain KD, Jones DR, Raison RL (2000) Characterization of mucosal and systemic Additional file 1: Table S1. Correlation (Pearson) value between each immune responses in rainbow trout (Oncorhynchus mykiss) using surface studied parameters in anterior intestine, age of tiger grouper (AGE), gap plasmon resonance. Fish Shellfish Immunol 10:651–666 between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Canan B, Do Nascimento WS, Da Silva NB, Chellappa S (2012) Morphohistology of number of goblet cells (NGC), number of villi (NOV), number of lymphoid the digestive tract of the damsel fish Stegastes fuscus (Osteichthyes: cells (NLC) and thickness of muscle (TOM). Pomacentridae). The Scientific World Journal Article ID 787316:9, Additional file 2: Table S2. Correlation (Pearson) value between each doi:10.1100/2012/787316 studied parameters in mid intestine, age of tiger grouper (AGE), gap Clements KD, Raubenheimer D (2006) Feeding and Nutrition. In: Evans DH, between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Claiborne JB (eds) The physiology of fishes. Taylor and Francis Group, Boca number of goblet cells (NGC), number of villi (NOV), number of lymphoid Raton, FL, USA cells (NLC) and thickness of muscle (TOM). Cole AM, Weis P, Diamond G (1997) Isolation and characterization of pleurocidin, Additional file 3: Table S3. Correlation (Pearson) value between each an antimicrobial peptide in the skin secretions of winter flounder. J Biol studied parameters in posterior intestine, age of tiger grouper (AGE), gap Chem 272:12008–12013 between villi (GBV), thickness of lamina propria (TLP), length of villi (LOV), Collie NL (1985) Intestinal nutrient transport in coho salmon (Oncorhynchus number of goblet cells (NGC), number of villi (NOV), number of lymphoid kisutch) and the effects of development, starvation, and seawater adaptation. cells (NLC) and thickness of muscle (TOM). J Comp Physiol B 156:163–174 Concha MI, Molina S, Oyarzun C, Villanueva J, Amthauer R (2003) Local expression of apolipoprotein A-I gene and a possible role for HDL in primary Competing interests defence in the carp skin. Fish Shellfish Immunol 14:259–73 The authors declare that they have no competing interests. Dabrowski K (1990) Absorption of ascorbic acid andascorbic sulfate and ascorbate metabolism in stomachless fish, Common carp. J Comp Physiol B Authors’ contribution 160:549–561 MFN has carried out the research work and analyzed the date, MZS outlined Dalmo RA, Ingebrightsen K, Bogwald J (1997) Non-specific defense mechanisms the research design and supervised the experimental trial, MZAB and MEAW in fish, with particular reference to the reticuloendothelial system (RES). J Fish helped revised the project and read the manuscript. All authors read and Dis 20:241–273 approved the final manuscript. De Silva SS (1998) Tropical Mariculture: Current Status and Prospects. Trop Mariculture 1:1–16 Ellis AE (2001) The immunology of teleosts. In: Roberts RJ (ed) Fish Pathology. WB Acknowledgement Saunders, London, pp 133–150 The authors wish to thank the Marine Aquaculture Breeding Technology Ezeasor DN, Stokoe WM (1981) Light and electron microscopic studies on the Centre, Tanjung Demong, Terengganu, Malaysia for fish supply and facilities absorptive cells of the intestine ceca and rectum of the adult rainbow trout, for conducting this study. We thank Ahmad Baihaqi Othman and Dr. Siti Salmo gairdneri. J Fish Biol 18:527–544 Zahrah Abdullah for their technical assistance and advice. Federal Agricultural Marketing Authority of Malaysia (2013) National Agro-food Author details Policy 2010–2020., Accessed on October 26, 2013 at http://www.fama.gov. Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor my/en/dasar-agromakanan-negara-2011-2020-dan-#.UpSxQo0Vg3Y 43400, Malaysia. Marine Finfish Production and Research Centre, FRI Tanjung Fernandes JMO, Kemp GD, Smith VJ (2004) Two novel muramidases from skin Demong, Besut, Terengganu 22200, Malaysia. Institute of Marine mucosa of rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu, B 138:53–64 Terengganu 21030, Malaysia. Ferraris RP, Yasharpour S, Lloyd KC, Mirzayan R, Diamond JM (1990) Luminal glucose concentrations in the gut under normal conditions. Am J Physiol Received: 3 October 2013 Accepted: 7 November 2013 259:G822–G837 Published: 15 November 2013 Firdaus-Nawi M, Sabri MY, Hanan Y, Siti-Zahrah A, Zamri-Saad S (2012) Efficacy of feed-based adjuvant vaccine against Streptococcus agalactiae in Oreo- chromis spp. in Malaysia. 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Thai Fisheries Gazette, Thai National AGRIS Centre (Thailand), Main Library, Kasertsart University, PO Box 1084, Kasertsart Chatuchak, Bangkok 10903, Thailand doi:10.1186/2193-1801-2-611 Cite this article as: Firdaus-Nawi et al.: Histological assessments of intestinal immuno-morphology of tiger grouper juvenile, Epinephelus fuscoguttatus. SpringerPlus 2013 2:611. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com

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