Animal Science Journal (2016) 87, 67–75
doi: 10.1111/asj.12399
ORIGINAL ARTICLE Influence of weaning age on the villous height and disaccharidase activities in the porcine small intestine Takamitsu TSUKAHARA,1,2 Ryo INOUE,1 Masako NAKATANI,1 Kikuto FUKUTA,3 Eriko KISHINO,4 Tetsuya ITO4 and Kazunari USHIDA1 1
Laboratory of Animal Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto Institute of Nutrition & Pathology, Kyoto, 3Technical Center, Toyohashi Feed Mills, Shinsiro and 4Ensuiko Sugar Refining, Carbohydrate Research Laboratory, Yokohama, Japan 2
ABSTRACT Body weight gain after weaning is correlated with villous height and disaccharidase activity. This evidence suggests that the maintenance of the small intestinal structure and function after weaning is important for the growth of piglets. We demonstrated that the influence of weaning age was obtained by disaccharidase activities and villous height in eight sections of the porcine small intestine. Therefore, we designed three weaning ages (weaned at the ages of 14, 21 or 28 days) and the piglets were slaughtered after 7 or 14 days post-weaning. The remaining suckling piglets were slaughtered at the age of 1, 7, 14, 21 and 28 days. Four piglets were slaughtered at each event; therefore, 44 piglets were used in this study. Villous height and disaccharidase activities were measured in each section of the small intestine. Early weaning such as that at 14 days had severe influence on villous and disaccharidase activities. In particular, weaning of 14-day-olds did not result in maltase activity at least 2 weeks post-weaning. Accordingly, the weaning age of crossbred piglets is recommended to be at least 21 days or more on the basis of villous height and disaccharidase activity analyses.
Key words: disaccharidase activity, pig, small intestine, villous height, weaning age.
INTRODUCTION The epithelial cells lining the intestinal villi consist of columnar enterocytes, also recognized as absorptive cells. The apical membrane of enterocytes with microvilli is also named the brush-border membrane. The brush-border membrane of enterocytes includes various channels and nutrient transporters to absorb various nutrients (Zhang & Xu 2003). Digestive enzymes such as disaccharidases and peptidases are secreted from the brush-border membrane to facilitate nutrient absorption. Among disaccharidases, the activities of maltase (MA) and sucrase in the brushborder membrane of the small intestine increase with age, whereas the activity of lactase (LA) attenuates as a result of diet conversion, for example weaning (Kidder & Manners 1980). Weaning is a stressful event for neonatal pigs. Weaning afflicts suckling piglets with physiological, social, environmental and dietary stresses that interfere with gut development (Lalles et al. 2004). Consequently, the morphology and function of the small intestine is severely disturbed after weaning (Wijtten et al. 2011). The small intestine and its mucosa lose © 2015 Japanese Society of Animal Science
20%–30% of their relative weight during the first 2 days after weaning, whereas regeneration requires 5–10 days for full recovery (Lalles et al. 2004). In particular, early weaning influences the small intestine, for example by causing villous shortening (Pluske 2001; Tsukahara et al. 2010) and reduction of disaccharidase activity (Hampson 1986). Villous shortening is assumed to impair the digestive ability of the small intestine, thereby inducing malabsorption and diarrhea in weaning piglets (van Beers-Schreurs et al. 1998; Marion et al. 2002). The sections of the small intestine are also important factors for considering the disaccharidase activities in pig (Dahlqvist 1961). In our previous study, we reported that remarkable LA activity was observed in the ileum of suckling piglets (Tsukahara et al. 2013). We also described that villous
Correspondence: Takamitsu Tsukahara, Laboratory of Animal Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan. (Email: [email protected]) Received 19 November 2014; accepted for publication 26 January 2015.
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shortening by early weaning was detected at the proximal jejunum and ileum but not at other sections (Tsukahara et al. 2010). In the present study, we demonstrated that the influence of weaning age was obtained by disaccharidase (LA and MA) activities and villous height in eight sections of the porcine small intestine. Therefore, we designed three weaning ages (weaned at the ages of 14, 21 or 28 days) and the piglets were slaughtered after 7 or 14 days post-weaning. The remaining suckling piglets were slaughtered at the ages of 1, 7, 14, 21 and 28 days. We discuss a suitable weaning age for suckling piglets according to the abovementioned parameters.
MATERIALS AND METHODS Animals One-day-old suckling piglets (S1), 7-day-old suckling piglets (S7), 14-day-old suckling piglets (S14), 21-day-old suckling piglets (S21), 28-day-old suckling piglets (S28), 21-day-old pigs weaned at the age of 14 days (W14p7), 28-day-old pigs weaned at the age of 14 days (W14p14), 28-day-old pigs weaned at the age of 21 days (W21p7), 35-day-old pigs weaned at the age of 21 days (W21p14), 35-day-old pigs weaned at the age of 28 days (W28p7), and 42-day-old pigs weaned at the age of 28 days (W28p14) were used. The slaughter schedule and mean body weight of piglets are listed in Table 1. All animals were crossbred (Landrace × Large white × Duroc) piglets and were bred at the Technical Center of Toyohashi Feed Mills (Shinshiro, Aichi, Japan). Suckling piglets were merely fed breast milk. Weaning piglets received a commercial diet for weaning (JustOne Sprout; Toyohashi Feed Mills, Aichi, Japan). The nutrient composition of this diet (g/kg) was as follows: crude protein, 214; crude fat, 75; crude fiber, 3; and crude ash, 60. All diets and water were fed ad libitum. The experimental animals were handled in accordance with the guidelines for animal studies of the Experimental Animal Committee of Kyoto Prefectural University.
Dissection, collection and analyses of the small intestine Dissection and collection procedures of the small intestine were the same as those previously described (Tsukahara et al. 2013). In brief, each pig was euthanized by exsanguination under general anesthesia with intraperitoneal injections of sodium pentobarbital (Somnopentyl; Kyoritsu, Tokyo, Japan). After a midline incision, the entire intestine was removed and the small intestine was separated. The length of
Table 1
the small intestine was measured and it was divided into eight sections of equal length. The sections were sequentially numbered from SI-1 to SI-8 from the proximal to the distal end. The SI-1 section was considered as the duodenum and proximal jejunum, the SI-2 to SI-4 sections were considered as the jejunum, the SI-5 and SI-6 sections were considered as the jejunum and ileum complex, and the SI-7 and SI-8 sections were considered as the ileum (Tsukahara et al. 2013). The middle segment of each section was collected for the histological study. The proximal segment of each section was used for the enzymatic study. Each segment of the small intestine used for the histological study was longitudinally incised and fixed in a 10% (v/v) phosphate-buffered formalin solution. A section was reserved for the enzymatic study. The mucosa of the small intestinal segment was scraped using a clean glass slide and stored at −80°C until analysis. Villous height was measured from hematoxylin–eosin (HE) staining preparations by light microscopy, as previously described (Tsukahara et al. 2010). The enzymatic study from the porcine intestinal mucosa was the same as that previously described (Tsukahara et al. 2013).
Statistical analyses Differences in each variable among slaughter points such as S1, S7, S14, S21, S28, W14p7, W14p14, W21p7, W21p14, W28p7 and W28p14 were analyzed by one-way analysis of variance (ANOVA). When significant differences were detected, the least significant difference post hoc comparison was used. Differences among means were considered significant at P < 0.05. All data were analyzed using STATCEL (OMS, Saitama, Japan), an add-in application for Microsoft Excel (Seattle, WA, USA).
RESULTS Length of the small intestine (Fig. 1) The length of the small intestine was approximately 400 cm at S1, and gradual growth was observed from S1 to S28. Significant growth was observed from S1 to S7 and from S21 to S28. In contrast, weaning influence was obscure at any weaning age. Significant growth was observed from W14p7 to W14p14, S21 to W21p7, W21p7 to W21p14 and S28 to W28p7.
Villous height in the small intestine (Fig. 2) Villous height was approximately 1.1 to 1.2 mm at S1 in all sections of the small intestine, and gradual shortening was observed from S1 to S28. Shortening
The slaughter schedule and abbreviations in this study
Piglet
Suckling 14-day-old weaned 21-day-old weaned 28-day-old weaned
Age 1 day
7 day
14 day
21 day
28 day
35 day
42 day
S1: 1.8 – – –
S7: 3.7 – – –
S14: 5.3 – – –
S21: 7.0 W14p7: 6.2 – –
S28: 10.4 W14p14: 8.9 W21p7: 9.0 –
– – W21p14: 13.1 W28p7: 12.4
– – W28p14: 15.9
Four piglets were slaughtered at the respective periods. Columns include abbreviations with mean body weight (kg) of four piglets.
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Figure 1 Length (cm) of the small intestine from 1-day-old to 42-day-old pigs. The mean values of the suckling piglets are indicated with closed squares; those of the piglets weaned at the age of 28 days are indicated with open circles; those of the piglets weaned at the age of 21 days are indicated with open triangles; and those of the piglets weaned at the age of 14 days are indicated with open rhombuses. The error bars represent standard errors. Asterisks represent that the difference was significant compared with the previous sampling point.
was typically observed at the distal sections of the small intestine, such as SI-5, SI-6, SI-7 and SI-8. Villous height in these distal sections of the small intestine in S28 piglets was approximately half of that in S1 piglets. Significant shortening of villous height was observed from S14 to S21 at SI-4, S14 to S21 at SI-5, S14 to S21 at SI-6, S1 to S7 and S21 to S28 at SI-7, and S1 to S7 at SI-8. Significant elongation of villous height was only detected from S7 to S14 at SI-7. The proximal sections of the small intestine, such as SI-1, SI-2, SI-3 and SI-4, were remarkably influenced by weaning. Villous shortening was typically observed in piglets weaned at the age of 14 days or 21 days. Significant shortening was observed from: S14 to W14p7 at SI-1; S14 to W14p7, S21 to W21p7 and S28 to W28p7 at SI-2; S14 to W14p7 and S21 to W21p7 at SI-3; S14 to W14p7, W14p7 to W14p14 and S21 to W21p7 at SI-4; S14 to W14p7 and S21 to W21p7 at SI-6; and S14 to W14p7 and S21 to W21p7 at SI-7. Significant elongation was observed from W21p7 to W21p14 at SI-6. Animal Science Journal (2016) 87, 67–75
LA activity in the small intestine (Fig. 3) LA activity was gradually activated from S1 to S14 at SI-1 to SI-7. Further activation was observed from S14 to S21 at SI-2, SI-3 and SI-7. The highest activity was approximately 400 U in the proximal sections of the small intestine, whereas 500–600 U of LA activity was obtained at SI-5 and SI-6. Different transition of LA activity was observed at SI-8. Attenuation was typically observed from S1 to S7, reversal activation was then observed from S7 to S21, and absolute attenuation was observed from S21 to S28. In addition, compared with other sections of the small intestine, the lowest LA activity was detected in SI-8. A rapid decline was detected after the peak of LA activity even in the suckling piglets; thus, LA activity was diminished in all sections of the small intestine in S28 piglets. Significant activation was detected from S1 to S7 at SI-3 and from S7 to S14 at SI-5. Significant attenuation was detected from S14 to S21 at SI-1, S21 to S28 at SI-2, S21 to S28 at SI-3, S21 to S28 at SI-4, S14 to S21 and S21 to S28 at SI-5, and S21 to S28 at SI-7. © 2015 Japanese Society of Animal Science
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Figure 2 Villous height (μm) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. The sections were numbered SI-1 to SI-8 from the proximal to the distal sections of the small intestine. For other details, please see Figure 1.
© 2015 Japanese Society of Animal Science
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Figure 3 Lactase activity (U) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. For details, please see Figures 1 and 2.
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Early weaning (weaned at the age of 14 days) induced rapid reduction of LA activity at SI-2 to SI-8. The weaning influence was limited in piglets weaned at the age of 21 days because LA activity was already attenuated even in suckling piglets at the age of 21 days. Therefore, a minimum influence was observed in piglets weaned at the age of 28 days. Significant attenuation was observed from: S14 to W14p7 at SI-1; S14 to W14p7 and S21 to W21p7 at SI-2; S14 to W14p7 and S21 to W21p7 at SI-3; S14 to W14p7 and S21 to W21p7 at SI-4; S14 to W14p7 and S21 to W21p7 at SI-5; S14 to W14p7 and S21 to W21p7 at SI-6; S14 to W14p7 and S21 to W21p7 at SI-7; and S14 to W14p7 and S21 to W21p7 at SI-8. Significant activation was detected from W21p7 to W21p14 at SI-2.
MA activity in the small intestine (Fig. 4) MA activity was also gradually activated by age in the suckling stage. The activation was observed from S1 to S21 at SI-1 to SI-7. The highest activity was approximately 400 U in the proximal sections and SI-5, whereas it was 900 U and 500 U at SI-6 and SI-7, respectively. The lowest MA activity was detected in SI-8; 200 U or less of MA activity was obtained throughout the suckling stage. Significant activation was detected from: S1 to S7 at SI-1; S14 to S21 at SI-2; S1 to S7 and S14 to S21 at SI-3; S1 to S7 and S14 to S21 at SI-4; S1 to S7 at SI-5; S1 to S7 at SI-6; and S14 to S21 at SI-7. Significant attenuation was detected from S21 to S28 at SI-3 and from S21 to S28 at SI-4. Early weaning (weaned at the age of 14 days) induced the attenuation of MA activity at the age of 21 days (7 days post-weaning) in all sections of the small intestine. The attenuation persisted at the age of 28 days (14 days post-weaning), except for SI-1. Weaning influence was limited in piglets weaned at the age of 21 days; the attenuation of MA activity was observed at the age of 28 days (7 days post-weaning), whereas the recovery of MA activity was observed in the proximal sections of the small intestine (SI-1 to SI-4). A minimum influence was observed for weaning at the age of 28 days. Significant attenuation was observed from: S14 to W14p7 and S21 to W21p7 at SI-1; S21 to W21p7 at SI-2; S21 to W21p7 at SI-3; S14 to W14p7, S21 to W21p7 and S28 to W28p7 at SI-4; S21 to W21p7 at SI-5; S21 to W21p7 at SI-6; and S21 to W21p7 at SI-7. Significant activation was detected from W28p7 to W28p14 at SI-4.
DISCUSSION The length of the small intestine was gradually elongated depending on the age in the suckling period (Fig. 1). In contrast, villous height was shortened depending on the age in the suckling period (Fig. 2). Among the disaccharidase activities, LA and MA activities were gradually activated during the suckling © 2015 Japanese Society of Animal Science
period. The highest activity was obtained at the age of 14 or 21 days; disaccharidase activity was attenuated at the age of 21 or 28 days despite continuously suckling (Figs 3,4). Neonatal development of the gastrointestinal system is a very dynamic process. After birth, the length of the villi quickly increases by the large volume of blood distributed into the intestinal mucosa (Zabielski et al. 2008). It is also known that the length of the small intestine gradually elongates (Adeola & King 2006). Villous height in the small intestine was shortened from 7-day-old to 21-day-old breast-fed piglets (Marion et al. 2005). Therefore, the suckling piglets in this study showed normal development at least in the small intestine. Weaning induced the elongation of the small intestine (Zijlstra et al. 1996), whereas villous height was shortened (Pluske 2001; Tsukahara et al. 2011). Early weaning such as that at 14 days has been found to induce serious villous atrophy in comparison with later weaning such as that at 28 days (Pluske 2001). In this study, weaning age was designed at 14 days, 21 days and 28 days, and a similar tendency of villous atrophy was observed at 7 days post-weaning (Fig. 2). In contrast, the recovery of villous atrophy from 7 days to 14 days post-weaning was affected by the weaning age. The recovery of villous atrophy was not observed in piglets weaned at the age of 14 days; the villus was still shortened at least 14 days post-weaning (Fig. 2). In contrast, the villus was elongated from 7 days to 14 days post-weaning in piglets weaned at the age of 21 days or 28 days. Remarkable villous atrophy was observed at the proximal (SI-1 to SI-4) and distal (SI-6 and SI-7) sections by early weaning (Fig. 2). Sectionspecific villous atrophy by weaning was also observed in a murine model (Tsukahara et al. 2010) and porcine study (Kelly et al. 1991a), and the atrophy sections showed a similar tendency in these studies. LA activity quickly develops in the small intestine after birth and is maintained during suckling (Manners & Stevens 1972). Kelly et al. (1991a) reported that LA and MA activities in the small intestine were activated (1.32- and 2.26-fold, respectively) from the ages of 14 days to 22 days in suckling piglets. However, another researcher reported that jejunal LA activity was attenuated, whereas MA activity was activated from the ages of 7 days to 21 days in suckling piglets (Marion et al. 2005); this result was similar to that of our present study. A part of the experimental design of Kelly’s report was obscure, and there was limited discussion on these differences. The breed of pigs may differ between Kelly’s study and our study because the mean body weight of 14-day-old piglets was smaller in Kelly’s study than in our study (4.5 kg vs. 5.3 kg). Moreover, LA activity continuously declines regardless of suckling following further development after the age of 3 weeks (Kelly et al. 1991b). This tendency was similar to that of our present study. Our previous study Animal Science Journal (2016) 87, 67–75
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Figure 4 Maltase activity (U) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. For details, please see Figures 1 and 2.
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demonstrated that remarkable LA activity was observed in distal sections of the small intestine, such as SI-7 in 21-day-old suckling piglets (Tsukahara et al. 2013). Although a similar tendency was observed, bimodal activities were detected in proximal (SI-2) and distal (SI-7) sections of S21 piglets in this study. Bimodal activity was also reported by Kelly et al. (1991b) and Manners and Stevens (1972). Manners and Stevens (1972) also described that single or double peaks of LA activity were observed in 21-day-old suckling piglets, and these differences were explained by litter individuality. Therefore, the difference in LA activity (single or double peaks) between our previous and the present study may explain litter individuality. The highest LA activity among the suckling piglets was detected in SI-5 and SI-6 of S14 (Fig. 3). These results suggest that the distal section of the small intestine is one of the major sites for lactose digestion during the suckling stage. MA activity was activated from the age of 1 day to 21 days during the suckling period, whereas the activity declined from the age of 21 days to 28 days despite suckling (Fig. 4). During the suckling period, lactose is the main component of dietary carbohydrates for pigs; MA activity was rarely considered in the previous reports. In contrast, in our previous study, we found that MA activity temporarily declined during the weaning period, while it recovered as the piglets grew (Tsukahara et al. 2013). Therefore, the decline of MA activity in S28 piglets may be a developmental process of the small intestine. Reduced LA activity followed by increased MA activity during weaning has been consistently reported (Manners & Stevens 1972; Kelly et al. 1991a; Pluske 2001; Marion et al. 2005). The composition of dietary carbohydrates for pigs changes tremendously from the suckling to the growing stage; the carbohydrates in sow’s milk are mainly lactose, whereas the diet offered to weaning and growing pigs is mainly composed of starch. Therefore, the intestinal tract of pigs has to modify its morphological structure and digestive enzymes in response to the dietary changes (Bach Knudsen & Jorgensen 2001). In contrast, temporary reduction of MA activity was observed for weaning at the age of 21 days or more, and minimal values were observed at the end of the first week post-weaning (Hampson 1986; Pluske et al. 1996). In this context, weaning seriously influenced the disaccharidase activities among piglets weaned at the age of 14 days (Figs 3,4). In particular, piglets weaned at the age of 14 days did not develop MA activity at least 2 weeks post-weaning (Fig. 4). Some researchers demonstrated that early weaning (at the age of 14 days or younger) induced no alteration or even activation of MA activity during the immediate post-weaning period (Kelly et al. 1991a; Tang et al. 1999; Marion et al. 2005). These results may explain that MA activity was not developed before S14; however, MA was activated since © 2015 Japanese Society of Animal Science
S14; therefore, there may be no alteration in MA activity and no alteration in early weaning piglets, as mentioned in this study. The highest MA activity was observed in S21, and the apparent reduction seemed remarkable in piglets weaned at the age of 21-day piglets. MA activity was attenuated from S21 to S28, and weaning influence on MA activity seemed moderate in piglets weaned at the age of 28 days. Within the first 4 postnatal weeks, the piglet weight is developed by >5-fold, with the gastrointestinal organs growing faster than many other organs of the body (Zabielski et al. 2008). Body weight gain after weaning is therefore correlated with villous height (Pluske et al. 1996). This evidence suggests that the maintenance of the small intestinal structure and function after weaning is important for the growth of piglets. Our results demonstrated that early weaning such as that at the age of 14 days caused severe villous atrophy, because early weaning induced adverse effects on the neonatal piglets. Our recent metagenomic study of the ileal mucosa demonstrated upregulation of the cell cycle pathway and downregulation of the cell adhesion pathway from S14 to S21 piglets (Inoue R, Tsukahara T, Nakatani M, Okutani M, Nishibayashi R, Ogawa S, Harayama T, Nagino T , Hatanaka H, Fukuta K, Ushida K, Imke M, Kelly D. unpubl. data). These data suggested that epithelial cells in the small intestine were remodeling not only their structure but also their function from suckling to weaning. Accordingly, the weaning age of crossbred piglets is recommended to be at least 21 days or more on the basis of villous height and disaccharidase activity analyses.
ACKNOWLEDGMENTS This experiment was funded by the Grant-in-Aid for Young Scientists (No. 23780269) from the Japan Society for the Promotion of Science. The authors thank Messrs. M. Nishikawa and K. Suzuki and Ms. N. Matsukawa of the Kyoto Institute of Nutrition & Pathology, and Messrs. H. Hatanaka and T. Nagino, and Mses. T. Harayama and Y. Shimizu of Kyoto Prefectural University for their technical assistance. The authors would like to thank Enago for the English language review.
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continuous nutrition after weaning. Animal Science 62, 131–144. Tang M, Laarveld B, Van Kessel AG, Hamilton DL, Estrada A, Patience JF. 1999. Effect of segregated early weaning on postweaning small intestinal development in pigs. Journal of Animal Science 77, 3191–3200. Tsukahara T, Inoue R, Yamada K, Yajima T. 2010. A mouse model study for the villous atrophy of the early weaning piglets. Journal of Veterinary Medical Science 72, 241–244. Tsukahara T, Kishino E, Inoue R, Nakanishi N, Nakayama K, Ito T, Ushida K. 2013. Correlation between villous height and the disaccharidase activity in the small intestine of piglets from nursing to growing. Animal Science Journal 84, 54–59. Tsukahara T, Yoshida Y, Tsushima T, Watanabe T, Matsubara N, Inoue R, Ushida K. 2011. Evaluation of the heat-killed and dried cell preparation of Enterococcus faecalis against villous atrophy in early-weaned mice and pigs. Animal Science Journal 82, 302–306. van Beers-Schreurs HMG, Nabuurs MJA, Vellenga L, Kalsbeek-van der Valk HJ, Wensing T, Breukink HJ. 1998. Weaning and the weanling diet influence the villous height and crypt depth in the small intestine of pigs and alter the concentrations of short-chain fatty acids in the large intestine and blood. Journal of Nutrition 128, 947– 953. Wijtten PJA, van der Meulen J, Verstegen MWA. 2011. Intestinal barrier function and absorption in pigs after weaning: a review. British Journal of Nutrition 105, 967–981. Zabielski R, Godlewski MM, Guilloteau P. 2008. Control of development of gastrointestinal system in neonates. Journal of Physiology and Pharmacology 59, 35–54. Zhang YQ, Xu RJ. 2003. Anatomy and histology of the gastrointestinal tract. In: Xu RJ, Cranwell PD (eds), The Neonatal Pig, pp. 1–30. Nottingham University Press, Nottingham. Zijlstra RT, Whang KY, Easter RA, Odle J. 1996. Effect of feeding a milk replacer to early-weaned pigs on growth, body composition, and small intestinal morphology, compared with suckled littermates. Journal of Animal Science 74, 2948–2959.
© 2015 Japanese Society of Animal Science
doi: 10.1111/asj.12399
ORIGINAL ARTICLE Influence of weaning age on the villous height and disaccharidase activities in the porcine small intestine Takamitsu TSUKAHARA,1,2 Ryo INOUE,1 Masako NAKATANI,1 Kikuto FUKUTA,3 Eriko KISHINO,4 Tetsuya ITO4 and Kazunari USHIDA1 1
Laboratory of Animal Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto Institute of Nutrition & Pathology, Kyoto, 3Technical Center, Toyohashi Feed Mills, Shinsiro and 4Ensuiko Sugar Refining, Carbohydrate Research Laboratory, Yokohama, Japan 2
ABSTRACT Body weight gain after weaning is correlated with villous height and disaccharidase activity. This evidence suggests that the maintenance of the small intestinal structure and function after weaning is important for the growth of piglets. We demonstrated that the influence of weaning age was obtained by disaccharidase activities and villous height in eight sections of the porcine small intestine. Therefore, we designed three weaning ages (weaned at the ages of 14, 21 or 28 days) and the piglets were slaughtered after 7 or 14 days post-weaning. The remaining suckling piglets were slaughtered at the age of 1, 7, 14, 21 and 28 days. Four piglets were slaughtered at each event; therefore, 44 piglets were used in this study. Villous height and disaccharidase activities were measured in each section of the small intestine. Early weaning such as that at 14 days had severe influence on villous and disaccharidase activities. In particular, weaning of 14-day-olds did not result in maltase activity at least 2 weeks post-weaning. Accordingly, the weaning age of crossbred piglets is recommended to be at least 21 days or more on the basis of villous height and disaccharidase activity analyses.
Key words: disaccharidase activity, pig, small intestine, villous height, weaning age.
INTRODUCTION The epithelial cells lining the intestinal villi consist of columnar enterocytes, also recognized as absorptive cells. The apical membrane of enterocytes with microvilli is also named the brush-border membrane. The brush-border membrane of enterocytes includes various channels and nutrient transporters to absorb various nutrients (Zhang & Xu 2003). Digestive enzymes such as disaccharidases and peptidases are secreted from the brush-border membrane to facilitate nutrient absorption. Among disaccharidases, the activities of maltase (MA) and sucrase in the brushborder membrane of the small intestine increase with age, whereas the activity of lactase (LA) attenuates as a result of diet conversion, for example weaning (Kidder & Manners 1980). Weaning is a stressful event for neonatal pigs. Weaning afflicts suckling piglets with physiological, social, environmental and dietary stresses that interfere with gut development (Lalles et al. 2004). Consequently, the morphology and function of the small intestine is severely disturbed after weaning (Wijtten et al. 2011). The small intestine and its mucosa lose © 2015 Japanese Society of Animal Science
20%–30% of their relative weight during the first 2 days after weaning, whereas regeneration requires 5–10 days for full recovery (Lalles et al. 2004). In particular, early weaning influences the small intestine, for example by causing villous shortening (Pluske 2001; Tsukahara et al. 2010) and reduction of disaccharidase activity (Hampson 1986). Villous shortening is assumed to impair the digestive ability of the small intestine, thereby inducing malabsorption and diarrhea in weaning piglets (van Beers-Schreurs et al. 1998; Marion et al. 2002). The sections of the small intestine are also important factors for considering the disaccharidase activities in pig (Dahlqvist 1961). In our previous study, we reported that remarkable LA activity was observed in the ileum of suckling piglets (Tsukahara et al. 2013). We also described that villous
Correspondence: Takamitsu Tsukahara, Laboratory of Animal Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan. (Email: [email protected]) Received 19 November 2014; accepted for publication 26 January 2015.
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shortening by early weaning was detected at the proximal jejunum and ileum but not at other sections (Tsukahara et al. 2010). In the present study, we demonstrated that the influence of weaning age was obtained by disaccharidase (LA and MA) activities and villous height in eight sections of the porcine small intestine. Therefore, we designed three weaning ages (weaned at the ages of 14, 21 or 28 days) and the piglets were slaughtered after 7 or 14 days post-weaning. The remaining suckling piglets were slaughtered at the ages of 1, 7, 14, 21 and 28 days. We discuss a suitable weaning age for suckling piglets according to the abovementioned parameters.
MATERIALS AND METHODS Animals One-day-old suckling piglets (S1), 7-day-old suckling piglets (S7), 14-day-old suckling piglets (S14), 21-day-old suckling piglets (S21), 28-day-old suckling piglets (S28), 21-day-old pigs weaned at the age of 14 days (W14p7), 28-day-old pigs weaned at the age of 14 days (W14p14), 28-day-old pigs weaned at the age of 21 days (W21p7), 35-day-old pigs weaned at the age of 21 days (W21p14), 35-day-old pigs weaned at the age of 28 days (W28p7), and 42-day-old pigs weaned at the age of 28 days (W28p14) were used. The slaughter schedule and mean body weight of piglets are listed in Table 1. All animals were crossbred (Landrace × Large white × Duroc) piglets and were bred at the Technical Center of Toyohashi Feed Mills (Shinshiro, Aichi, Japan). Suckling piglets were merely fed breast milk. Weaning piglets received a commercial diet for weaning (JustOne Sprout; Toyohashi Feed Mills, Aichi, Japan). The nutrient composition of this diet (g/kg) was as follows: crude protein, 214; crude fat, 75; crude fiber, 3; and crude ash, 60. All diets and water were fed ad libitum. The experimental animals were handled in accordance with the guidelines for animal studies of the Experimental Animal Committee of Kyoto Prefectural University.
Dissection, collection and analyses of the small intestine Dissection and collection procedures of the small intestine were the same as those previously described (Tsukahara et al. 2013). In brief, each pig was euthanized by exsanguination under general anesthesia with intraperitoneal injections of sodium pentobarbital (Somnopentyl; Kyoritsu, Tokyo, Japan). After a midline incision, the entire intestine was removed and the small intestine was separated. The length of
Table 1
the small intestine was measured and it was divided into eight sections of equal length. The sections were sequentially numbered from SI-1 to SI-8 from the proximal to the distal end. The SI-1 section was considered as the duodenum and proximal jejunum, the SI-2 to SI-4 sections were considered as the jejunum, the SI-5 and SI-6 sections were considered as the jejunum and ileum complex, and the SI-7 and SI-8 sections were considered as the ileum (Tsukahara et al. 2013). The middle segment of each section was collected for the histological study. The proximal segment of each section was used for the enzymatic study. Each segment of the small intestine used for the histological study was longitudinally incised and fixed in a 10% (v/v) phosphate-buffered formalin solution. A section was reserved for the enzymatic study. The mucosa of the small intestinal segment was scraped using a clean glass slide and stored at −80°C until analysis. Villous height was measured from hematoxylin–eosin (HE) staining preparations by light microscopy, as previously described (Tsukahara et al. 2010). The enzymatic study from the porcine intestinal mucosa was the same as that previously described (Tsukahara et al. 2013).
Statistical analyses Differences in each variable among slaughter points such as S1, S7, S14, S21, S28, W14p7, W14p14, W21p7, W21p14, W28p7 and W28p14 were analyzed by one-way analysis of variance (ANOVA). When significant differences were detected, the least significant difference post hoc comparison was used. Differences among means were considered significant at P < 0.05. All data were analyzed using STATCEL (OMS, Saitama, Japan), an add-in application for Microsoft Excel (Seattle, WA, USA).
RESULTS Length of the small intestine (Fig. 1) The length of the small intestine was approximately 400 cm at S1, and gradual growth was observed from S1 to S28. Significant growth was observed from S1 to S7 and from S21 to S28. In contrast, weaning influence was obscure at any weaning age. Significant growth was observed from W14p7 to W14p14, S21 to W21p7, W21p7 to W21p14 and S28 to W28p7.
Villous height in the small intestine (Fig. 2) Villous height was approximately 1.1 to 1.2 mm at S1 in all sections of the small intestine, and gradual shortening was observed from S1 to S28. Shortening
The slaughter schedule and abbreviations in this study
Piglet
Suckling 14-day-old weaned 21-day-old weaned 28-day-old weaned
Age 1 day
7 day
14 day
21 day
28 day
35 day
42 day
S1: 1.8 – – –
S7: 3.7 – – –
S14: 5.3 – – –
S21: 7.0 W14p7: 6.2 – –
S28: 10.4 W14p14: 8.9 W21p7: 9.0 –
– – W21p14: 13.1 W28p7: 12.4
– – W28p14: 15.9
Four piglets were slaughtered at the respective periods. Columns include abbreviations with mean body weight (kg) of four piglets.
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was typically observed at the distal sections of the small intestine, such as SI-5, SI-6, SI-7 and SI-8. Villous height in these distal sections of the small intestine in S28 piglets was approximately half of that in S1 piglets. Significant shortening of villous height was observed from S14 to S21 at SI-4, S14 to S21 at SI-5, S14 to S21 at SI-6, S1 to S7 and S21 to S28 at SI-7, and S1 to S7 at SI-8. Significant elongation of villous height was only detected from S7 to S14 at SI-7. The proximal sections of the small intestine, such as SI-1, SI-2, SI-3 and SI-4, were remarkably influenced by weaning. Villous shortening was typically observed in piglets weaned at the age of 14 days or 21 days. Significant shortening was observed from: S14 to W14p7 at SI-1; S14 to W14p7, S21 to W21p7 and S28 to W28p7 at SI-2; S14 to W14p7 and S21 to W21p7 at SI-3; S14 to W14p7, W14p7 to W14p14 and S21 to W21p7 at SI-4; S14 to W14p7 and S21 to W21p7 at SI-6; and S14 to W14p7 and S21 to W21p7 at SI-7. Significant elongation was observed from W21p7 to W21p14 at SI-6. Animal Science Journal (2016) 87, 67–75
LA activity in the small intestine (Fig. 3) LA activity was gradually activated from S1 to S14 at SI-1 to SI-7. Further activation was observed from S14 to S21 at SI-2, SI-3 and SI-7. The highest activity was approximately 400 U in the proximal sections of the small intestine, whereas 500–600 U of LA activity was obtained at SI-5 and SI-6. Different transition of LA activity was observed at SI-8. Attenuation was typically observed from S1 to S7, reversal activation was then observed from S7 to S21, and absolute attenuation was observed from S21 to S28. In addition, compared with other sections of the small intestine, the lowest LA activity was detected in SI-8. A rapid decline was detected after the peak of LA activity even in the suckling piglets; thus, LA activity was diminished in all sections of the small intestine in S28 piglets. Significant activation was detected from S1 to S7 at SI-3 and from S7 to S14 at SI-5. Significant attenuation was detected from S14 to S21 at SI-1, S21 to S28 at SI-2, S21 to S28 at SI-3, S21 to S28 at SI-4, S14 to S21 and S21 to S28 at SI-5, and S21 to S28 at SI-7. © 2015 Japanese Society of Animal Science
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Figure 2 Villous height (μm) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. The sections were numbered SI-1 to SI-8 from the proximal to the distal sections of the small intestine. For other details, please see Figure 1.
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Figure 3 Lactase activity (U) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. For details, please see Figures 1 and 2.
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Early weaning (weaned at the age of 14 days) induced rapid reduction of LA activity at SI-2 to SI-8. The weaning influence was limited in piglets weaned at the age of 21 days because LA activity was already attenuated even in suckling piglets at the age of 21 days. Therefore, a minimum influence was observed in piglets weaned at the age of 28 days. Significant attenuation was observed from: S14 to W14p7 at SI-1; S14 to W14p7 and S21 to W21p7 at SI-2; S14 to W14p7 and S21 to W21p7 at SI-3; S14 to W14p7 and S21 to W21p7 at SI-4; S14 to W14p7 and S21 to W21p7 at SI-5; S14 to W14p7 and S21 to W21p7 at SI-6; S14 to W14p7 and S21 to W21p7 at SI-7; and S14 to W14p7 and S21 to W21p7 at SI-8. Significant activation was detected from W21p7 to W21p14 at SI-2.
MA activity in the small intestine (Fig. 4) MA activity was also gradually activated by age in the suckling stage. The activation was observed from S1 to S21 at SI-1 to SI-7. The highest activity was approximately 400 U in the proximal sections and SI-5, whereas it was 900 U and 500 U at SI-6 and SI-7, respectively. The lowest MA activity was detected in SI-8; 200 U or less of MA activity was obtained throughout the suckling stage. Significant activation was detected from: S1 to S7 at SI-1; S14 to S21 at SI-2; S1 to S7 and S14 to S21 at SI-3; S1 to S7 and S14 to S21 at SI-4; S1 to S7 at SI-5; S1 to S7 at SI-6; and S14 to S21 at SI-7. Significant attenuation was detected from S21 to S28 at SI-3 and from S21 to S28 at SI-4. Early weaning (weaned at the age of 14 days) induced the attenuation of MA activity at the age of 21 days (7 days post-weaning) in all sections of the small intestine. The attenuation persisted at the age of 28 days (14 days post-weaning), except for SI-1. Weaning influence was limited in piglets weaned at the age of 21 days; the attenuation of MA activity was observed at the age of 28 days (7 days post-weaning), whereas the recovery of MA activity was observed in the proximal sections of the small intestine (SI-1 to SI-4). A minimum influence was observed for weaning at the age of 28 days. Significant attenuation was observed from: S14 to W14p7 and S21 to W21p7 at SI-1; S21 to W21p7 at SI-2; S21 to W21p7 at SI-3; S14 to W14p7, S21 to W21p7 and S28 to W28p7 at SI-4; S21 to W21p7 at SI-5; S21 to W21p7 at SI-6; and S21 to W21p7 at SI-7. Significant activation was detected from W28p7 to W28p14 at SI-4.
DISCUSSION The length of the small intestine was gradually elongated depending on the age in the suckling period (Fig. 1). In contrast, villous height was shortened depending on the age in the suckling period (Fig. 2). Among the disaccharidase activities, LA and MA activities were gradually activated during the suckling © 2015 Japanese Society of Animal Science
period. The highest activity was obtained at the age of 14 or 21 days; disaccharidase activity was attenuated at the age of 21 or 28 days despite continuously suckling (Figs 3,4). Neonatal development of the gastrointestinal system is a very dynamic process. After birth, the length of the villi quickly increases by the large volume of blood distributed into the intestinal mucosa (Zabielski et al. 2008). It is also known that the length of the small intestine gradually elongates (Adeola & King 2006). Villous height in the small intestine was shortened from 7-day-old to 21-day-old breast-fed piglets (Marion et al. 2005). Therefore, the suckling piglets in this study showed normal development at least in the small intestine. Weaning induced the elongation of the small intestine (Zijlstra et al. 1996), whereas villous height was shortened (Pluske 2001; Tsukahara et al. 2011). Early weaning such as that at 14 days has been found to induce serious villous atrophy in comparison with later weaning such as that at 28 days (Pluske 2001). In this study, weaning age was designed at 14 days, 21 days and 28 days, and a similar tendency of villous atrophy was observed at 7 days post-weaning (Fig. 2). In contrast, the recovery of villous atrophy from 7 days to 14 days post-weaning was affected by the weaning age. The recovery of villous atrophy was not observed in piglets weaned at the age of 14 days; the villus was still shortened at least 14 days post-weaning (Fig. 2). In contrast, the villus was elongated from 7 days to 14 days post-weaning in piglets weaned at the age of 21 days or 28 days. Remarkable villous atrophy was observed at the proximal (SI-1 to SI-4) and distal (SI-6 and SI-7) sections by early weaning (Fig. 2). Sectionspecific villous atrophy by weaning was also observed in a murine model (Tsukahara et al. 2010) and porcine study (Kelly et al. 1991a), and the atrophy sections showed a similar tendency in these studies. LA activity quickly develops in the small intestine after birth and is maintained during suckling (Manners & Stevens 1972). Kelly et al. (1991a) reported that LA and MA activities in the small intestine were activated (1.32- and 2.26-fold, respectively) from the ages of 14 days to 22 days in suckling piglets. However, another researcher reported that jejunal LA activity was attenuated, whereas MA activity was activated from the ages of 7 days to 21 days in suckling piglets (Marion et al. 2005); this result was similar to that of our present study. A part of the experimental design of Kelly’s report was obscure, and there was limited discussion on these differences. The breed of pigs may differ between Kelly’s study and our study because the mean body weight of 14-day-old piglets was smaller in Kelly’s study than in our study (4.5 kg vs. 5.3 kg). Moreover, LA activity continuously declines regardless of suckling following further development after the age of 3 weeks (Kelly et al. 1991b). This tendency was similar to that of our present study. Our previous study Animal Science Journal (2016) 87, 67–75
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Figure 4 Maltase activity (U) of eight sections in the small intestine from 1-day-old to 42-day-old pigs. For details, please see Figures 1 and 2.
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demonstrated that remarkable LA activity was observed in distal sections of the small intestine, such as SI-7 in 21-day-old suckling piglets (Tsukahara et al. 2013). Although a similar tendency was observed, bimodal activities were detected in proximal (SI-2) and distal (SI-7) sections of S21 piglets in this study. Bimodal activity was also reported by Kelly et al. (1991b) and Manners and Stevens (1972). Manners and Stevens (1972) also described that single or double peaks of LA activity were observed in 21-day-old suckling piglets, and these differences were explained by litter individuality. Therefore, the difference in LA activity (single or double peaks) between our previous and the present study may explain litter individuality. The highest LA activity among the suckling piglets was detected in SI-5 and SI-6 of S14 (Fig. 3). These results suggest that the distal section of the small intestine is one of the major sites for lactose digestion during the suckling stage. MA activity was activated from the age of 1 day to 21 days during the suckling period, whereas the activity declined from the age of 21 days to 28 days despite suckling (Fig. 4). During the suckling period, lactose is the main component of dietary carbohydrates for pigs; MA activity was rarely considered in the previous reports. In contrast, in our previous study, we found that MA activity temporarily declined during the weaning period, while it recovered as the piglets grew (Tsukahara et al. 2013). Therefore, the decline of MA activity in S28 piglets may be a developmental process of the small intestine. Reduced LA activity followed by increased MA activity during weaning has been consistently reported (Manners & Stevens 1972; Kelly et al. 1991a; Pluske 2001; Marion et al. 2005). The composition of dietary carbohydrates for pigs changes tremendously from the suckling to the growing stage; the carbohydrates in sow’s milk are mainly lactose, whereas the diet offered to weaning and growing pigs is mainly composed of starch. Therefore, the intestinal tract of pigs has to modify its morphological structure and digestive enzymes in response to the dietary changes (Bach Knudsen & Jorgensen 2001). In contrast, temporary reduction of MA activity was observed for weaning at the age of 21 days or more, and minimal values were observed at the end of the first week post-weaning (Hampson 1986; Pluske et al. 1996). In this context, weaning seriously influenced the disaccharidase activities among piglets weaned at the age of 14 days (Figs 3,4). In particular, piglets weaned at the age of 14 days did not develop MA activity at least 2 weeks post-weaning (Fig. 4). Some researchers demonstrated that early weaning (at the age of 14 days or younger) induced no alteration or even activation of MA activity during the immediate post-weaning period (Kelly et al. 1991a; Tang et al. 1999; Marion et al. 2005). These results may explain that MA activity was not developed before S14; however, MA was activated since © 2015 Japanese Society of Animal Science
S14; therefore, there may be no alteration in MA activity and no alteration in early weaning piglets, as mentioned in this study. The highest MA activity was observed in S21, and the apparent reduction seemed remarkable in piglets weaned at the age of 21-day piglets. MA activity was attenuated from S21 to S28, and weaning influence on MA activity seemed moderate in piglets weaned at the age of 28 days. Within the first 4 postnatal weeks, the piglet weight is developed by >5-fold, with the gastrointestinal organs growing faster than many other organs of the body (Zabielski et al. 2008). Body weight gain after weaning is therefore correlated with villous height (Pluske et al. 1996). This evidence suggests that the maintenance of the small intestinal structure and function after weaning is important for the growth of piglets. Our results demonstrated that early weaning such as that at the age of 14 days caused severe villous atrophy, because early weaning induced adverse effects on the neonatal piglets. Our recent metagenomic study of the ileal mucosa demonstrated upregulation of the cell cycle pathway and downregulation of the cell adhesion pathway from S14 to S21 piglets (Inoue R, Tsukahara T, Nakatani M, Okutani M, Nishibayashi R, Ogawa S, Harayama T, Nagino T , Hatanaka H, Fukuta K, Ushida K, Imke M, Kelly D. unpubl. data). These data suggested that epithelial cells in the small intestine were remodeling not only their structure but also their function from suckling to weaning. Accordingly, the weaning age of crossbred piglets is recommended to be at least 21 days or more on the basis of villous height and disaccharidase activity analyses.
ACKNOWLEDGMENTS This experiment was funded by the Grant-in-Aid for Young Scientists (No. 23780269) from the Japan Society for the Promotion of Science. The authors thank Messrs. M. Nishikawa and K. Suzuki and Ms. N. Matsukawa of the Kyoto Institute of Nutrition & Pathology, and Messrs. H. Hatanaka and T. Nagino, and Mses. T. Harayama and Y. Shimizu of Kyoto Prefectural University for their technical assistance. The authors would like to thank Enago for the English language review.
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