FULL PAPER Anatomy

Digestibility of and Pigeon Seed Meals and Morphological Intestinal Alterations in Pigs

Apichai MEKBUNGWAN1), Narin THONGWITTAYA1) and Koh-en YAMAUCHI2)*

1)Department of Animal Technology, Faculty of Agricultural Production, Maejo University, Sansai, Chiang Mai, 50290, and 2)Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken 761–0795, Japan

(Received 25 August 2003/Accepted 19 January 2004)

ABSTRACT. To compare the nutrient digestibility of soybean meal (SM) and pigeon pea seed meal (PM) as well as morphological intestinal alterations in piglets fed them, three pigs per group were randomly selected at the end of the feeding experiment for ten days. Growth performance was higher in the SM group than in the PM group (p<0.05). The digestibility of crude protein, crude and crude fiber was 80.6%, 23.6% and 52.4% in the SM group, while in the PM group, values of 49.8%, 23.6% and 43.2% were observed, respectively. Digestible energy was 3.26 kcal g-1 in SM and 3.17 kcal g-1 in PM. It was concluded that the digestibility of PM was lower than that of SM; almost half of the in PM was digested. Dietary treatments had no effect on length of each small intestinal segment and weight of visceral organs (small intestine, liver, heart, spleen, kidney, stomach and lung) except the decreased kidney weight in the PM group (p<0.05). The epithelial cells on the jejunal villi showed a dome-like shape in the SM group, but they were a flat shape in the PM group. The present digestion trial and histological intestinal data suggest that the intestinal digestive and absorptive functions are much more atrophied in the PM group than in the SM group, and demonstrate that histological intestinal alterations might be well related with the intestinal functions. KEY WORDS: digestibility, pigeon pea seed, soybean meal, swine. J. Vet. Med. Sci. 66(6): 627–633, 2004

Soybean meal (SM) is an important in the human The intestinal villi assume the role of digestion and diet, and also provides a large proportion of dietary absorption in uptake diets. The villus height showed a rapid protein in animal feed. The use of alternative legume seeds alteration after rearing under various conditions [10, 22, 25], is encouraged to supply a constant plant protein source in and the villus height and epithelial cells on the villus tip animal feed and to reduce dependence on SM imported to recovered immediately after refeeding in pigs [18]. The villi Thailand. Pigeon pea ( cajan L.) is a perennial of the small intestine as well as epithelial cells on the duode- legume indigenous to tropical and sub-tropical regions, and nal villi were histologically much more hypertrophied in pigeon pea seed meal (PM) has been studied widely from pigs fed SM than in those fed PM [19]. These studies sug- nutritional [20, 30] and growth performance [4, 7, 20] stand- gest that the histological intestinal alterations might be points. Although its use is limited by the higher content of directly related to intestinal function. Although numerous anti-nutritional factors such as trypsin and chymotrypsin studies dealing with histological intestinal alterations inhibitors than SM [2, 34], Ene-Obong [7] has reported that related to fed diets have been published, the direct relation- such anti-nutritional factors do not cause problems in ani- ship between histological intestinal alterations and intestinal mals based on in vitro protein digestibility. In feeding functions in the same animals has not thus far been studied. experiments, PM could substitute SM in diets for pig based As most of nutrients in PM were almost half content of those on growth performance [20]. However, a clear understand- in SM [20], SM and PM are thought to be the most suitable ing of the nutrient digestibility in PM is still lacking. For feed ingredients for judging the intestinal function. The sec- ruminants, PM could be substituted for SM in diets based on ond aim of this study was to establish the relationship its digestibility in sheep [4]. Compared with digestion trials between histological intestinal alterations and intestinal using herbivores, the use of omnivores is crucial due to functions more clearly. effects of feed ingredients other than PM on digestibility. In In this study, SM or PM was fed to growing pigs, and poultry, the metabolic energy of was established by growth performance, the nutrient digestibility of SM and feeding only one dietary component [6]. digest- PM, the intestinal length and weight of visceral organs as ibility of vetch was measured by force-feeding only one well as epithelial cells on the tip surface of jejunal villi were source of protein [8]. It was therefore concluded that an compared. ingredient-feeding method described by these authors would be the most suitable method as an initial experiment MATERIALS AND METHODS of the digestibility of PM in pigs. The first aim of this study was to establish the digestibility of SM and PM. Animals, diets, and feeding: SM was purchased from a commercial company (Porn Amnouy Thrup Co., Ltd., *CORRESPONDENCE TO: PROF. DR. YAMAUCHI, K., Laboratory of Ani- mal Science, Faculty of Agriculture, Kagawa University, Miki- Sukothai, Thailand). The pigeon pea harvested in Thailand cho, Kagawa-ken, 761–0795, Japan. was prepared by crushing with a hammermill until small 628 A. MEKBUNGWAN, N. THONGWITTAYA AND K-E YAMAUCHI

Table 1. Composition of control diet, soybean meal (SM) and Calculation of nutrient digestibility: Nutrient digestibility pigeon pea seed meal (PM) was calculated according to the method described by Pond, et al. [26] as follows. Items Control SM PM Ingredients, g kg-1 Apparent (nutrient intake – nutrient in feces) × 100 Maize 672.8 – – digestibility (%) = nutrient intake bran 50.0 – – Soybean meal (SM) 207.2 1000.0 – Pigeon pea seed meal (PM) – – 1000.0 In this experiment, for example, we calculated for the Fish meal 50.0 – – digestibility of gross energy; Dicalcium phosphate 15.0 – – Energy intake = GEi × Fi, Premix ( & )* 5.0 – – Energy in feces = GEe × Fe GEi = Gross energy in feed intake (kcal/g) -1 Chemical composition, g kg GEe = Gross enery in feces (kcal/g) Dry matter 881.5 870.8 882.3 Fi = Feed intake (g) Crude protein 181.6 450.1 232.5 Crude fat 72.1 49.5 19.4 Fe = Feces (g) Crude fiber 31.1 43.7 64.3 Ash 67.6 78.1 48.1 Digestibility of (GEi× Fi) – (GEe × Fe) = × 100 (%) 10.3 3.1 2.1 gross energy (GEi × Fi) 7.7 6.1 3.0 Gross anatomical protocol: Immediately after slaughter, Gross energy, kcal g-1 4.20 4.13 4.53 the abdominal cavity was opened along the midline, and * Premix supplies (per kg diet): Vitamin A (3,333 IU), VItamin D then the visceral organs were excised in their entirety. Sam- (667 IU), (0.33 mg), Vitamin K3 (0.66 mg), Vitamin B2 ples of each small intestinal segment were collected accord- (1.67 mg), Vitamin B12 (0.003 mg), Calcium pantothenate (6.67 mg), Cobalt (3.47 mg), (27.60 mg), Iodine (0.77 mg), ing to their serous surface structure: a duodenal sample was (18.47 mg), (50.00 mg), and Fe (60.00 mg). prepared from the stomach pylorus caudally to the duode- nal-jejunal junction at the cranial end of duodenocolic fold enough to pass through a 4-mm sieve. At 85-d of age, 15 (showing thicker and slight blood vessel network on the castrated male crossed pigs were kept in individual metabo- serous surface than that of jejunum, about 55 cm length), lism cages, and allotted to conventional diet (Table 1, con- and an ileal sample was prepared from the ileal-cecal ostium trol), SM or PM groups. The animals were given ad libitum rostrally to the jejunal-ileal junction at the attaching area of access to diets and water. SM and PM were supplemented iliocecal fold on ileum (showing larger but fewer square to the basal diet at 25% on day 1, 50% on day 2, 75% on day blood vessel network than that of the jejunum, about 200 cm 3 and 100% on day 4. Finally SM or PM was fed for seven length). The remaining middle section of the small intestine days for adaptation. At that point, the pigs (average body was the jejunum. Each part was cut, washed with 0.9% weight, 29.3 kg) were fed one of the experimental diets NaCl solution to remove the intestinal contents, and the including 1% chromium oxide (Cr2O3, green color) as a length and weight of each part was measured. Visceral marker for collecting colored feces for the following three organs such as the liver, heart, spleen, kidney, stomach and days to determine the digestibility of nutrients in the control lung were also weighed. diet, SM and PM. During the last three days, feed intake and Statistical analysis: Data regarding growth performance, body weight were measured, and marked feces were col- digestibility of nutrients, and length and weight of visceral lected twice a day. After measuring the weight of wet feces, organs were statistically analyzed by using one-way analy- it was dried and ground into powder. At the end of each sis of variance (ANOVA), and significant difference among experiment, three pigs per group (average body weight, 31.6 the treatments were determined with Duncan’s multiple kg) were slaughtered at the slaughter house in the Maejo range test using the Stat View® program (Abacus Concepts, University in Thailand for observations of visceral organs Inc., HULINKS, Inc., Tokyo, Japan). Differences were and epithelial cells on the jejunal villi. All experiments declared significant at p<0.05. were carried out according to the humane care guidelines for Scanning electron microscopic protocol: Immediately the care and use of laboratory animals established by the after excising the visceral organs, a 2 × 3 cm block of the Faculty of Agriculture of Kagawa University. jejunum was taken from the middle of the small intestine, The gross energy of the feces and diets was measured and slit longitudinally along the entire length of its non- using a ballistic bomb calorimeter (IKA-Analysentechnik mesenteric side. The intestinal contents were removed with GMBH, PO. Box 1240, Heitersheim D-79420, Germany), 0.01 M phosphate-buffered saline (pH 7.4), and samples and their chemical composition was analyzed at the Depart- were pinned flat to prevent curling and to fix them vertically ment of Animal Technology, Maejo University, Thailand with the mucosal face downwards in a mixture of 3% glut- (Table 1, see Mekbungwan et al. [20] for chemical compo- araldehyde and 4% paraformaldehyde in 0.1 M cacodylate sition, such as the essential amino acid content of PM and buffer (pH 7.4) at room temperature for 1 hr. The tissue SM). block was further cut into a 3 × 10 mm rectangle and fixed DIGESTIBILITY OF PIGEON PEA IN PIGS 629

Table 2. Growth performance of pigs fed a control diet, soybean meal (SM) or pigeon pea seed meal (PM) (n=5) Items Control SM PM P value Initial BW, kg 29.2 ± 0.40 29.6 ± 0.70 29.0 ± 0.70 0.80 Final BW, kg 31.3 ± 0.30 31.2 ± 0.30 29.9 ± 0.70 0.21 ADG, kg 0.69 ± 0.03a) 0.54 ± 0.03b) 0.28 ± 0.02c) <0.01 ADFi, kg 0.94 ± 0.03a) 0.60 ± 0.02b) 0.44 ± 0.04c) <0.01 BW gain kg/kg feed 0.73 ± 0.04b) 0.89 ± 0.02a) 0.62 ± 0.20c) 0.01 Values are means ± SEM. BW=Body weight. ADG=Average daily gain. ADFi=Average daily feed intake. a) b) c) Means in the same row followed by different superscript letters are significantly different (p<0.05).

Table 3. Digestibility of nutrients in a control diet, soybean meal (SM) and pigeon pea seed meal (PM) (n=5) Items Control SM PM P value Digestibility, % Dry matters 73.8 ± 1.8 75.6 ± 2.5 71.2 ± 3.4 0.52 Crude protein 64.2 ± 2.5b) 80.6 ± 2.3a) 49.8 ± 4.9c) <0.01 Crude fat 70.8 ± 2.9a) 23.6 ± 3.0b) 23.6 ± 2.7b) <0.01 Crude fiber 18.0 ± 2.9b) 52.4 ± 5.1a) 43.2 ± 4.2a) <0.01 Ash 27.0 ± 3.9 35.8 ± 2.9 40.2 ± 5.5 0.38 Calcium 46.4 ± 6.3 35.8 ± 3.2 38.4 ± 6.1 0.09 Phosphorus 47.2 ± 3.8 47.6 ± 4.8 31.2 ± 7.1 0.05 Gross energy 76 ± 1b) 79 ± 1a) 70 ± 4b) 0.01

Digestible energy, kcal g-1* 3.20 3.26 3.17 Values are means ± SEM. * Digestible energy is calculated from gross energy (Table 1) × digestion percentage of gross energy/100. a) b) c) Means in the same row followed by different superscript letters are significantly different (p<0.05). for a further 1 hr. The pieces were rinsed with 0.1 M fiber digestibility of SM and PM did not differ significantly cacodylate buffer (pH 7.4) and post-fixed with 1% osmium and was higher than that of the control diet (SM, PM > con- tetroxide in ice-cold buffer for 2 hr. The specimens were trol, p<0.05); SM tended to be higher than PM (SM > PM). dried in a critical-point drying apparatus (Hitachi HCP-1, The digestion percentage of gross energy was higher in the Hitachi Ltd., Tokyo, 100–8220 Japan). The dried speci- SM diet than in the control or PM diet (SM > control, PM, mens were coated with platinum (RMC-Eiko®RE vacuum p<0.05); in this regard, the control and the PM diets did not coater Eiko Engineering Co., Ltd., Tokyo, Japan), and differ significantly, but the PM tended to be lower than the observed with a scanning electron microscope (Hitachi S- control diet (control > PM). Therefore, the values of digest- 800, Hitachi Ltd., Tokyo, Japan). ible energy calculated from gross energy (Table 1) and digestion percentage of gross energy were highest in the SM RESULTS group but lowest in the PM group (SM > control > PM). In other nutrients, the significant difference was not observed Growth performance: Daily feed intake and body weight among each group. gain were significantly lower in the SM and PM groups Gross morphological observations: Intestinal length and compared with the control group (p<0.05), and in the PM weight of internal organs were similar amongst the groups group compared with the SM group (p<0.05) (control > SM (p>0.05), except for lower kidney weights in the PM group > PM, Table 2). Feed efficiency was highest in the SM than in the control and SM groups (p<0.05) (Table 4). group, followed by the control and PM groups (SM > con- Scanning electron microscopic observations: Among the trol > PM, p<0.05). conventional epithelial cells (large arrows) on the apical sur- Digestibility of nutrients: Table 3 shows the digestibility face of the control jejunal villi, some cells protuberated into of nutrients in control, SM and PM diets. Crude protein the intestinal lumen like a dome (small arrows) were digestibility was higher in the SM diet but lower in the PM observed (Fig. 1A). After feedings of SM, all epithelial than in the control (SM > control > PM, p<0.05). Crude fat cells developed to the dome-like cells, and cell outline digestibility of SM and PM was similar, and lower than that between each cell was clear (Fig. 1B). However, in the case of the control diet (control > SM = PM, p<0.05). Crude of the PM group, the epithelial cells became flat cells 630 A. MEKBUNGWAN, N. THONGWITTAYA AND K-E YAMAUCHI

Table 4. Intestinal length and weights of internal organs in pigs fed a control diet, soybean mean (SM) and pigeon pea seed mean (PM) (n=3) Item Control SM PM P value Length, cm Duodenum 51.0 ± 2.6 51.0 ± 5.5 66.0 ± 6.6 0.09 Jejunum 942.0 ± 57.8 1048.3 ± 3.4 1040.3 ± 6.5 0.46 Ileum 216.6 ± 11.8 244.6 ± 8.5 223.3 ± 10.6 0.18 Weight, g Duodenum 51.6 ± 6.1 54.3 ± 5.3 51.6 ± 4.1 0.92 Jejunum 675.0 ± 73.3 670.6 ± 40.9 617.6 ± 46.3 0.50 Ileum 157.6 ± 6.0 152.6 ± 5.4 128.6 ± 11.6 0.09 Liver 740.6 ± 59.4 715.3 ± 20.5 654.0 ± 4.1 0.46 Heart 127.6 ± 3.9 131.0 ± 8.0 130.0 ± 4.6 0.92 Spleen 58.6 ± 4.3 54.3 ± 4.6 54.3 ± 5.2 0.76 Kidney 145.6 ± 3.8a) 139.6 ± 0.8a) 115.3 ± 3.7b) 0.01 Stomach 252.6 ± 8.6 248.6 ± 16.7 226.0 ± 11.7 0.14 Lung 407.3 ± 13.9 361.3 ± 33.3 342.0 ± 3.0 0.15 Values are means ± SEM. a) b) Means in the same row followed by different superscript letters are significantly different (p<0.05).

(arrows), resulting in the smooth surface (Fig. 1C). formance in pigs fed the PM diet than pigs fed the SM diet. A consideration of the present results and the findings of DISCUSSION similar studies in other literature leads to the general conclu- sion that the digestive and absorptive functions would be Digestible energy and the digestibility of crude protein, lower in pigs fed PM than those fed SM. which are the most important factors for pig growth, showed Feed efficiency (body weight gain/feed intake) of SM and the highest values in the SM group, but lowest in the PM PM is 0.89 and 0.62, respectively. The price of SM and PM group. In growth performance, pigs fed SM showed lower is about 10.5 and 5.0 Baht/kg, respectively. When feed cost daily body-weight gain than those fed the control diet. is calculated based on price/feed efficiency, feed cost of SM However, the daily feed intake of the SM group was lower and PM becomes 11.79 and 8.06 Baht/kg of body weight than the control, resulting in higher feed efficiency in the gain, resulting in a lower feed cost per kg of body weight SM group than in the control. This result seems to correlate gain for PM than for SM. However, when the present to the higher crude protein content (450.1 g kg-1), crude pro- results, which show that almost half of the protein in PM tein digestibility (80.6%) and digestible energy (3.26 kcal g was digested, and the fact that SM is very expensive com- -1) of the SM diet than of the control (181.6 g kg-1, 64.2%, pared with PM, are taken into consideration, the effective and 3.20 kcal g-1, respectively). On the other hand, pigs fed utilization of PM will result in reduced pig feed costs. the PM diet showed the lowest values in these growth per- Weight of the intestine, liver and pancreas [12], and the formances, although the crude protein of the PM diet (232.5 stomach and large intestine [27] was unaffected by the high g kg-1) was higher than the control (181.6 g kg-1). This fiber diet. However, this result disagrees with another study might be due to the lower digestibility of nutrients in the PM [11, 13] which reported an increased visceral weight in diet than the control and SM diets, and by the fact that in the response to a high fiber intake. Jin et al. [12] discussed that PM diet, the content of most nutrients, such as essential such a decreased result due to fiber would be affected by amino acids, were almost half of those in the SM diet [20]. longer feeding periods than the two weeks of his study. Our Furthermore, the decreased growth performance of the PM results, showing no changes in visceral organs except the group seems to be affected by anti-nutritional factors such kidney, might be due to the short ten-day experimentation as trypsin and chymotrypsin inhibitors in raw PM [9, 28]. period. However, the kidney weight decreased after feed- The trypsin and chymotrypsin inhibitors (4.8 and 2.7 mg g-1 ings of PM. As a high protein diet induced increased rela- respectively) were in higher concentrations in the PM diet tive liver and kidney weights in experiments of up to 48 than in the SM (0.26 and 0.00 mg g-1, respectively) [2]. The days [1], the present decreased kidney weight is thought to trypsin inhibitor activity of PM (26 unit mg -1) was 10 times be related to the lower value of crude protein in PM than in that of SM [34], resulting in the decreased growth rate, feed SM, and the low digestibility of crude protein due to anti- intake and feed efficiency in pigs fed the PM diet [2]. The nutritional factors in PM. present decreased growth performance of PM group might It has been reported that the number of epithelial cells per be induced by anti-nutritional factors in raw PM, because villus and the villus surface area measured directly on a the raw PM was only crushing. In addition, other factors scanning electron microscopic micrograph correlated well such as differences in amino acid balance, protein/energy with the villus height and the number of epithelial cells of ratio, etc., would also have influenced the lower growth per- the same villus measured on the histological sections under DIGESTIBILITY OF PIGEON PEA IN PIGS 631

Fig. 1. Apical surface of jejunal villus in pig fed a control diet (A; large arrows, conven- tional cells; small arrows, dome-like cells), soybean meal (SM) (B) or pigeon pea seed meal (PM) (C; arrows, flat cells). Epithelial cells of the control group develop to dome- like cells in SM group, but become flat cells in PM group. A~C = scale bar, 10 µm. a light microscope [23]. From the fact that light micro- hypertrophied and found in the activated function of the scopic parameters such as intestinal villus height, cell area intestine, while flat cells are atrophied and seen in the not- and cell mitosis were decreased after fasting, but quickly as-activated intestinal function from a feeding experimental recovered after refeeding [18, 29, 31], the higher values of standpoint [18, 31]. However, intestinal functions such as the light microscopic parameters might be hypertrophied absorption for PM have not been actually measured in pigs and related to the activated function of the intestine. Dome- fed PM until now. Our final goal was to demonstrate like epithelial cells on the tips of villi are also reported to be whether or not the intestinal function is much more acti- 632 A. MEKBUNGWAN, N. THONGWITTAYA AND K-E YAMAUCHI vated in pigs fed SM than in those fed PM, namely whether present histological and physiological intestinal data in the or not histological intestinal alteration is related to intestinal same pigs demonstrate that the histological intestinal alter- function. We have reported that the trypsin inhibition rate ations might be well related to the intestinal functions. was decreased from 99.15% in raw PM to 54.31% in heated Although traditionally assessments of nutritional value of PM, resulting in much more improved growth performance fed diets has been evaluated using nutritional-physiological and higher light microscopic parameters in the pigs fed methods such as nutrient digestibility, the histological intes- heated PM than those fed raw PM; dome-like cells were tinal alterations might also be a useful index by which to found on the apical surface of the duodenal villi [16]. In the make an assessment of the nutritional value of feed and of present pigs fed SM, higher values of the growth perfor- intestinal function. mance and nutrient digestibility than those fed PM were In conclusion, the growth performance and nutrient observed. 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