| 行政院農業委員會-台灣農業故事館 | ||
 動物也吃健康食品 |
發布日期：95/11/14 | |
| 預防醫學風潮日盛，健康食品當紅，各式各樣新開發的健康食品讓消費者趨之若騖。但是，你知道嗎？動物也有健康食品喔！尤其是在農委會明令畜產養殖業者逐年減少使用抗生素飼料添加物後，動物用健康食品的研發愈來愈興盛，需求逐步增加，絲毫不輸給人類所使用的健康食品！
歐盟從2006年起，已經全面禁止畜禽養殖業者在飼料 農委會畜牧處牧場管理科科長林俊臣表示，養殖戶對抗生素的依賴其實是心理問題，並非技術問題。業者如果能體認到飼養管理是一切的根本，做好養殖場的安全衛生管理工作，讓家畜禽在一個良好健康的生長環境中成長，則生長促進用抗生素的使用就沒有那樣大的需要。 除了做好畜禽牧場的飼養管理，排除使用促進生長用抗生素之後，以台灣高溫多濕、高度密集養殖的現實下，如何尋找非藥物性的代替品，以確保家畜禽在生長過程中能安全健康地長大，成為重要的課題。這也是為什麼近年來不論是農委會或試驗研究單位都積極投入研發綠色飼料添加物的原因。 所謂綠色飼料添加物，廣義來講，指的就是無污染、無殘留、抗疾病和能促進生長的天然添加物，近年來先後已開發的種類包括微生物製劑、酵素製劑、酸化劑、調味劑、中草藥製劑和純天然萃取物等。
那麼，目前眾多研發出來的綠色飼料添加物劑中，較廣泛被使用的有哪些？動科所應用動物組副研究員廖震元指出，主要是益生菌、酸化劑和中草藥等，其中中草藥多為複方產品。 除了研發各項新的綠色飼料添加物劑外，如何利用生物技術，讓這些添加物能夠發揮最大的效用，也是研究人員積極努力的。例如，活菌進入消化道後，還未到達腸道前，大多會被胃酸消滅，效果有限。近年來，動科所就研發出動物用微膠囊化生菌劑的關鍵技術，並成功技轉給廠商，讓益生菌能以微膠囊包覆的方式進入動物體內，發揮最大作用。 廖震元說，儘管隨著潮流和政策法令的制訂，綠色飼料添加物的需求量日增，但這方面的商品幾乎都是仰賴進口品，國內廠商自行研發量產的很少。 林俊臣指出，綠色飼料添加物基本上是一種概念，生產出來的商品要令人接受，推廣給更多農民使用，一定要結合畜禽加工品的自創品牌，才有誘因和利基。 以台灣第一個生產過程全程記錄，並且受動科所控管的台東關山毛豬產銷班所生產的「晶鑽豬」為例，廖震元表示，「晶鑽豬」具有低用藥、無污染、無殘留、具產銷履歷等特色，即便售價是一般豬肉的兩倍，一樣供不應求，創造出亮麗的銷售佳績。因為賣得好，業者就會持續採用低用藥、使用綠色飼料添加物的作法，形成良性的循環。其他像「自然豬」、「安心豚」的品牌，也是同樣的道理。唯有結合品牌，國產益生菌等綠色飼料添加物的發展才有更廣闊的空間。 |
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Category: News
中使用促進生長用抗生素。因應歐盟的腳步以及安全、自然農業的發展趨勢，農委會動植物防疫檢疫局從民國89年起刪減安巴素等含藥物飼料添加物，並且自民國94年起分階段逐年禁止製造、輸入和使用含抗生素的飼料添加物。到2007年7月1日後，可以作為含藥物飼料添加物的抗生素品目只剩下14項。這三年內已被禁止不能再使用的10項抗生素包括「硫汰徽素」、「林可徽素」、「觀徽素」、「純徽素」、「配尼西林」、「新徽素」、「枯草菌素」、「可利斯汀」、「氯四環徽素」和「羥四環徽素 」。
台灣動物科技研究所副所長顏宏達指出，微生物製劑就是生菌劑，又可稱為益生菌，一般較熟悉者如乳酸菌和酵母菌等，作用是剌激有益微生物在畜禽的胃腸道內生長，可加強畜禽的腸道免疫力，並抑制病原菌的生長。以人類為例，許多人主張多食用優酪乳或含乳酸菌的健康食品，目的就在於健胃整腸、幫助消化，在動物身上也是一樣的道理。農委會畜牧處長黃英豪則以「這就好比給動物吃健康食品一樣」，比喻益生菌對畜禽養殖的作用。
顏宏達表示，有生產益生菌的國內廠商目前約有六、七家，確實不多。廖震元認為，這與國內廠商大多屬於中小企業型態，很難有大筆資金和人力投注在相關研發工作有關。研發出動物用微膠囊化生菌劑關鍵技術的動科所動物醫學組研究員廖朝暐則指出，國內要發展益生菌的市場，一定要與製藥界合作，不能單只靠農業界。猪料中禁用氧化锌的可能性
来源：国际畜牧网独家编译 2016年12月16日 点击：550
在英国，英国国家养猪协会（NPA）对欧洲药品管理局兽药产品委员会向兽药理事会做出的停止对含氧化锌兽药产品的营销许可的提议表示惊讶和失望。
英国国家养猪协会高级政策顾问Georgina Crayford在致兽药理事会的一封信中表示，“当然，使用氧化锌的环境风险是要考虑的一个重要因素，但是此前欧洲药品管理局兽药产品委员会所有的评估都认为氧化锌的益处大于对环境造成的威胁，所以可以想象我们对他们在未给出清晰解释前提下180度的态度转折的惊讶。”“欧洲药品管理局兽药产品委员会的评估显然忽略了一点，就是在禁用氧化锌之后由于抗菌药使用的增加导致的耐药性的风险。”
Crayford补充说，禁止使用氧化锌对猪只进行口腔处理会导致断奶后腹泻的增加，会对仔猪的动物福利产生不良影响，从而“严重影响猪业减少抗生素使用的能力。”
锌的环境评估：没有迫切影响
欧洲食品安全局饲料评审工作组此前对动物饲料中锌对环境的风险进行过评估。
饲料评审工作组在一份相关报道中称，“经过评估，使用锌化合物不会对农业土壤造成迫切影响。但是，潜在的环境顾虑是它会通过排放和溢流而进到地表水，其中酸性的砂质土壤最易受到影响。”
饲料评审工作组补充说，近期采用的饲料最大锌含量的提议会极大地降低含锌添加剂对环境造成的风险。
在此建议下，2016年7月，欧盟引进了动物饲料中锌使用的新规，包括降低鱼料中犊牛代乳料中锌的最大水平，而猪料中锌的水平不变（150 mg/kg）。
【国际畜牧网独家编译】
欧洲拟在猪料中禁用氧化锌
来源：国际畜牧网独家编译 2016年12月16日 点击：1699
欧洲药品管理局（EMA）称，用氧化锌防止猪只腹泻所带来的环境风险要大于其带来的益处。
欧洲药品管理局兽药产品委员会上周的决定将欧盟猪饲料中禁用氧化锌的进程又向前推进了一步。兽药产品委员会建议未来停止对含氧化锌兽药产品的营销许可，同时撤回对当前含有这种成分产品的营销许可。
考虑到使用氧化锌产品对环境带来的潜在影响以及耐药细菌的普遍性的增加，法国和荷兰将此问题提交给欧洲药品管理局兽药产品委员会。
兽药产品委员会达成的共识是，“氧化锌用以预防猪只腹泻的益处未及其对环境造成的危险。”
在抗生素耐药性方面，委员会则称使用氧化锌与耐药性共现的风险确实存在，但是这种风险无法量化。
高鋅飼料猛於虎！
1989年，丹麥科學家Poulsen首次提出，在斷奶仔豬飼料中添加藥理劑量的氧化鋅可顯降低斷奶後的腹瀉率。在當時，這被稱為二十世紀豬營養研究的里程碑式的發現之一。在之後接近三十年的實踐更是證明，此方法不僅有效，而且非常經濟，幾乎成為國內外飼料行業解決仔豬腹瀉的一種最常用的手段。但是很快，我們又發現使用氧化鋅特別是過量使用，會給生豬、人類以及整個環境帶來危害。比如長期使用藥理劑量的氧化鋅會降低生豬的採食量，從而抑制其生長；再比如高劑量的氧化鋅會導致糞便中鋅的大量排放，對人類的環境造成影響。
一、具體來看，氧化鋅的使用，特別是高鋅的危害猛於虎
1、 重金屬污染
由於部分廠家的氧化鋅質量控制不嚴格，很可能會受到鎘、砷、鉛等的污染，若添加到仔豬飼料中，將使得生豬機體受到鎘、砷、鉛等的污染，可能在屠宰時仍舊能檢測到它們的存在。
2、營養拮抗
高劑量的鋅會導致生豬機體產生過量的金屬硫蛋白，在腸道內可與銅元素結合，可能會導致腸道內的銅出現缺乏的問題。並且，高劑量的氧化鋅還會降低植酸酶的作用，使得生豬出現磷缺乏的症狀。同時也有研究證明，高劑量的氧化鋅還會中和一定數量的有機酸等酸從而降低有機酸的作用。
3、鋅有毒性，長期添加氧化鋅，將對生豬的健康產生負面作用，肉眼觀察到的結果為，皮膚蒼白，毛長等。
4、當日糧鋅濃度不超過150ppm時，通過豬糞富集到土壤中的鋅每年不會超過3000ug/kg乾物質。每噸飼料中添加3kg的氧化鋅，那麼豬一生排除的鋅元素會增加30%，這將增加豬場糞污處理以及環境消耗的負擔。
5、使用高劑量的氧化鋅後，高鋅在使一些病原菌對鋅具有抗性的同時，還會加速這些病原菌對抗生素產生耐藥性。
二、因此，許多國家開始立法，限制甚至禁止高劑量氧化鋅的使用。
2005年，歐盟便禁止氧化鋅在飼料中的添加，只能用作於獸藥處方使用；2009年，我國飼料添加劑安全使用規範規定仔豬斷奶後前2周配合飼料中氧化鋅形式的鋅的添加量不超過2250 mg/kg，其他階段的用量為43-120mg/kg。然而，為保證短期的效果，我國眾多飼料企業卻一直都採用的高鋅的配方，置生豬長期的健康與人類的環境於不顧。比如2016年7月18日農業部公布的嚴重不合格的飼料產品中，67家企業中有16家企業（其中不乏眾多國內大企業）的鋅含量超標，甚至有企業鋅含量超過國家標準的10倍以上。
可以說，高鋅、高銅的危害比瘦肉精更嚴重。在如此嚴峻的背景下，尋找氧化鋅的替代品無疑成為國內外飼料行業研究的重要課題。
ZnO is harmful!
Potentially harmful for the environment
The Committee for Medicinal Products for Veterinary Use (CVMP), part of the European Medicines Agency (EMA), scrutinised the feed additive earlier this month after it had been pointed out by countries like France and the Netherlands that ZnO could potentially harm the environment.
The committee concluded that ‘overall the benefit-risk balance for the product’ is negative, “as the benefits of zinc oxide for the prevention of diarrhoea in pigs do not outweigh the risks for the environment.”
ZnO and antibiotic-resistance
In addition, the use of ZnO is also said to be related to the increase of prevalence of antibiotic-resistant bacteria. The committee estimated that ‘at the present time, that risk is not quantifiable’.
Currently, the CVMP’s conclusion is only a recommendation and will take some time to take effect. The European Commission will have to make a final decision and prior to doing so, many stakeholders will be heard.
The characterization of butyrate transport across pig and human colonic luminal membrane
Corresponding author S. P. Shirazi-Beechey: Epithelial Function and Development Group, Department of Veterinary PreClinical Sciences, The University of Liverpool, Brownlow Hill and Crown Street, Liverpool L69 3BX, UK. Email: spsb@liverpool.ac.uk
Abstract
- 1Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [14C]butyrate.
- 2The activity of cysteine-sensitive alkaline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins, markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV were free from contamination by other cellular organelles and basolateral membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein.
- 3The transport of butyrate into the luminal membrane vesicles was enhanced 5-fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperature dependent, and was stimulated in the presence of an outward-directed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration showed saturation kinetics with an apparent Km value of 14.8 ± 3.6 mM and a Vmax of 54 ± 14 nmol min−1 (mg protein)−1.
- 4Butyrate transport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvate and L-lactate). Butyrate uptake was inhibited by several cysteine group modifying reagents such as p-chloromercuribenzosulphonic acid (pCMBS), p-chloromercuribenzoate (pCMB), mersalyl acid and HgCl2, but not by the stilbene anion exchange inhibitors, 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS) and 4,4′-dinitrostilbene-2,2′-disulphonate (SITS).
- 5The described properties of butyrate transport across the luminal pole of the colon suggest the involvement of a carrier protein, in the form of a pH-activated anion exchange process. The transporter is distinct from the erythrocyte band-3 type anion exchanger and may belong to the monocarboxylate-type transport proteins (MCT1).
http://onlinelibrary.wiley.com/doi/10.1111/j.1469-7793.1998.819bs.x/full
Is “Enteric” Necessary for Butyrate? 丁酸盐所谓的“肠溶”是个伪命题！
The following article is from the internet and for your kind reference and comments:
外界对丁酸盐原粉存在两种误解：
1）丁酸盐如果没用脂肪包被的话过不了胃部!
2）由于后肠道是碱性环境，所以丁酸易被解离， 以阴离子形式存在， 而丁酸根阴离子是没有效果的，因无法被吸收，也无杀菌能力!
他们的理由是：
1）丁酸盐在胃部被溶解，而丁酸透过胃壁被吸收掉了。
2）有机酸在不同酸碱值下被解离的情形如下：
| 解离/非解离 | ||||
| 有机酸种类 | pKa | pH 4 | pH 6 | pH 7 |
| 甲酸 | 3.75 | 1.8 : 1 | 178 : 1 | 1778 : 1 |
| 乳酸 | 3.83 | 1.5 : 1 | 148 : 1 | 1479 : 1 |
| 苯甲酸 | 4.19 | 0.6 : 1 | 65 : 1 | 646 : 1 |
| 丁酸 | 4.86 | 0.1 : 1 | 15 : 1 | 138 : 1 |
| 丙酸 | 4.88 | 0.1 : 1 | 13 : 1 | 132 : 1 |
酸性越强，丁酸越不易解离，大部分以完整丁酸形式存在 (解离/非解离 = 0.1 / 1)。
碱性越强，丁酸越易解离，大部分以丁酸根阴离子形式存在 (解离/非解离 = 138 / 1)。
而在后肠道中碱性较强 (pH > 7)，丁酸根阴离子 / 丁酸比例会大于 138 / 1. 所以他们宣称丁酸盐到达不了后肠道。
事实如此吗？
事实1：
早在1990年Galfi and Bokori 添加0.17%丁酸盐原粉于日粮中，喂给从7 – 102公斤的猪只，回肠绒毛长度增长及盲肠隐窝深度加深。
事实2：
2006年Manzanilla等人指出添加0.3%丁酸盐原粉可增加空肠绒毛长度及加深隐窝深度。回肠隐窝深度也加深了。结肠的杯形细胞数目也增加了。
以上2个事实证明丁酸盐原粉是可过胃，也可到达前后肠道。
那丁酸盐原粉是如何过胃到达前肠道及后肠道呢?
A、丁酸盐在胃部解离释放出钙或钠阳离子及丁酸根阴离子。胃部是酸性环境富含氢离子(H+)。氢离子又与丁酸根阴离子结合成为完整的丁酸被运送到肠道。
B、
1、丁酸以自动非离子扩散模式被吸收:
多年以来人们一直以为短链脂肪酸在结肠吸收是只以非离子扩散(non-ionic diffusion)的方式进入上皮细胞。
2、丁酸根阴离子以碳酸氢根离子转换的方式被吸收:
其实丁酸根阴离子还可在上皮细胞顶膜(apical membrane)及基底膜(basolateral membrane)透过HCO3– 交换(碳酸氢根离子转换的方式) 进入上皮细胞， 达到丁酸吸收的目的，此过程在结肠膜内的空泡(vesicle)中被证实。
3、丁酸根阴离子也以其他短链脂肪酸浓度的增加的方式被吸收:
Charney等人于1998年在远端结肠中更发现， 纵然在碱性的环境下(pH 7.4)，其他短链脂肪酸浓度的提高也有助丁酸根阴离子吸收，进入结肠上皮细胞内。并强调这种机制是不受酸碱值影响，也就是说短链脂肪酸浓度越高， 则丁酸根阴离子吸收度越高。
C、Smith, D. J. 等人于2012年(J. Agric. Food Chem. 60:3151-3157)以肉小鸡做试验，用碳14标定丁酸钙原粉中的丁酸，发现原粉丁酸钙在盲肠、结肠及泄殖腔释放的量在进食后2， 4，8及12小时分别为0.3%，0.2%，0.1% 及0.4%。 脂肪保护的丁酸钙在盲肠、 结肠及泄殖腔释放的量在进食后2， 4， 8及12小时分别为0.8%，0.4%， 0.2%及0.4%。
以上打破原粉到达不了后肠道的谣言!!
Butyrate: Short-chain fatty acids Absorption
Short-chain fatty acids and medium-chain fatty acids are primarily absorbed through the portal vein during lipid digestion,[6] while long-chain fatty acids are packed into chylomicrons and enter lymphatic capillaries, and enter the blood first at the subclavian vein.
https://en.wikipedia.org/wiki/Short-chain_fatty_acid
Kuksis, Arnis (2000). “Biochemistry of Glycerolipids and Formation of Chylomicrons”. In Christophe, Armand B.; DeVriese, Stephanie. Fat Digestion and Absorption. The American Oil Chemists Society. p. 163. ISBN 189399712X. Retrieved December 21, 2012.
Butyrate: Feeding the Gut and Beyond for Animal Health
James Pierce, MS, PhD; Nutriad Inc. Elgin, IL, USA – Maja Marien, DVM, PhD; Tim Goossens, PhD; Nutriad International NV Dendermonde, Belgium
Introduction
In North America, there has been a tremendous body of research in recent years to find the ideal product or program to replace or reduce the use of antibiotics in livestock and poultry production. This is driven by the legislation in the EU and the pressure the health-care system has placed on the use of sub-therapeutic levels of antibiotics for growth promotion. There have been many arguments made regarding the validity of such claims, but ultimately, the consumer will decide what they are willing to accept and pay for accordingly.
With the projected increase in the human population as well as the rise in income over the next 20 years, especially in BRIC countries, there will be a concomitant increase in demand for animal products. The demands to increase the safety and efficiency of food production has become a major focus globally. In recent years, many molecules and additives have been evaluated for their ability to improve animal health, growth, efficiency, and reproduction. The products evaluated have included: prebiotics, probiotics, botanical products, acids (inorganic, organic, short and medium chain fatty acids), chelated minerals, and enzymes.
In order to evaluate these compounds, one should aim at understanding as much as possible about their safety, efficacy, and mode of action. If we are primarily looking to replace antibiotic growth promoters, we should first evaluate the effect on digestion and gut health. The intestine is the largest organ in the body and thus involves a complex system in which several factors influence the final outcome. In the intestine, three major components (the mucosal barrier, the composition of the microbiota and the local immune system) provide defensive measures against different pathogens through permanent contact and communication with each other. Feed additives may interact with host cells (intestinal cells, immune cells), with the host’s microbiota, or with pathogens impairing the normal intestinal function.
Butyrate – Proposed modes of action
Butyrate is somewhat unique among its biological functions when compared with other short chain fatty acids (SCFA). The (SCFA) constitute a group of molecules that contain from one to seven carbon atoms and which exist as straight or branched-chain compounds: predominant SCFAs are acetic, propionic and butyric acid. Because SCFA are weak acids with a pK of ≤4.8 and the pH of the gastrointestinal tract (GIT) is nearly neutral, 90-99% of the SCFA are present in the GIT as anions rather than free acids.
Among the SCFA, butyric acid has received particular attention. Butyric acid is available in the salt form of Na, K, Mg or Ca. The advantage of salts over free acids is that they are generally odorless and easier to handle in the feed manufacturing process owing to their solid and less volatile form. For the purpose of the present article, the term ‘butyrate’ is used interchangeably for the acid, the salt and the anion forms.
Fig 1. Absorption of n-butyrate in the large intestine and subsequent metabolism. Butyrate transport with monocarboxylate transporters (MCT) are saturable and coupled with H+ transport. Butyrate is recognized by G-protein receptors (GPR41, GPR43). (Adapted from Guiloteau et al., 2010).
There are several potential fates of butyrate once consumed by the animal. Butyrate may function as a ligand for transmembrane receptors, as a modulator of gene activity, and as a direct energy source for cellular metabolism via B-oxidation. Several cell types, many of which associated with the digestive tract, have been described to be receptive to one or more of these biological effector functions, which explains the extensive range of biochemical effects that have been documented to be mediated by butyrate (Fig 1).
When butyrate is present in the blood stream or in the proximal parts of the intestinal tract, it induces the production of host defense peptides (Guilloteau et al., 2009). These peptides stimulate the development and repair of the intestinal tract through an increase in cell proliferation (Bartholome et al., 2004). Recently it has been shown that butyrate, when present in blood, stimulates a peptide that increases the absorption of glucose from the intestine. Indications that a similar mode of action can be expected in poultry, is shown by Hu and Guo (2007), who found an increased development of the villi when sodium-butyrate was added to the diet.
Butyrate also has been shown to stimulate several functions in the lower part of the intestinal tract. Studies have identified specific G-protein-coupled receptors, specifically GPR 41 and GPR 43, on gut epithelial cells in the epithelium of particularly the ileum, caeca and colon (Le Poul et al., 2003). When butyrate is attached to these receptors the production of several different peptides is stimulated (Cox et al., 2009, Tazoe et al., 2008). Some of these peptides have a positive effect on the development of the immune system and improve the functioning of the immune system in case of a health challenge (Cox et al., 2009). Other peptides have been shown to optimize gut motility, by reducing the rate of feed passage (Tazoe et al., 2008). In poultry, the emptying of the feed out of the gizzard into the small intestine is slowed down. Thus, it seems that butyrate is inducing a similar effect to passage rate as coarse particles such as oyster shell.
Indications that butyrate also stimulates the immune system in poultry were obtained by Leeson et al. (2005) in that, birds previously fed butyrate showed more ability to withstand against the stress of coccidial challenge at 21 d of age. Weber (2008) found when pigs where challenged with Escherichia coli lipopolysaccharide (LPS), sodium butyrate increased the magnitude of the cortisol response and increased skeletal muscle IL-6 mRNA expression, also indicating that dietary butyrate affects the response to inflammatory stimuli.
In summary, beneficial effects include, among others, stimulation of digestive enzyme production, enhanced development of intestinal villi, reduction of acute inflammatory responses, increased GIT retention time, inhibition of cancer cell growth and the secretion of host defense peptides (Guilloteau et al., 2012). Apart from effects in eukaryotic host cells, butyrate is also described to have an impact on the activity of prokaryotes residing in the animal’s GIT. For example, it has been shown to affect the colonization of Salmonella and Campylobacter and to influence the composition of the gut microbiota (see below).
Butyrate – Experimental results
Mucosal barrier
Dietary supplementation of butyrate has been shown to support enteric development and intestinal health of neonatal animals. At weaning, the small intestine of the piglet generally undergoes a decreased capacity of absorption that is associated with a marked reduction in villous height and crypt depth. These changes are accompanied with a decreased feed intake and poor growth (Piva et al., 2002; Pluske et al., 1996). Butyrate stimulates epithelial cell proliferation resulting in a larger absorptive surface, leading to improved feed utilization. Furthermore, butyrate in the weaner diet preserves villus length and thereby helps to maintain feed intake. Effect of butyrate on gut morphology is of great biological value to the weaning period when the weight of the small and large intestine increases three times faster than that of the whole body mass.
The following trial results (Ferket et al., 2010) were obtained in broilers, but do show the principle of the impact of butyrate supplementation on intestinal development and growth performance in young animals. Commercial broilers were randomly assigned to 32 floor pens containing 30 birds each and provided feed and water ad libitum until 49 days. Starter feed (pellet-crumbled) treatments consisting of 3 dietary supplementation levels of coated butyrate (0.0, 0.015, 0.03, and 0.06% butyrate) were subjected to 8 replicate pens per treatment from 1 – 14 days. Subsequently, all birds were fed common grower and finisher diets in pelleted form. Body weight (BW) and feed intake was determined at 7, 14, 21, 42, and 49 days and feed/gain (FCR) calculated. At 3, 8, and 14 days, 4 birds/treatment were sampled for gut histology evaluation. BW at 14 days increased linearly (p<0.01) as the level of butyrate increased (457 g vs. 470 g for 0 vs. 0.06% butyrate), but no effects on 1-14 days FCR were observed. Histomorphometic analysis correlated with early treatment effects on BW (see Fig 2A and 2B). The positive starter feed treatment effects was observed throughout the experiment, with 0.015% butyrate resulting in a 3% and 2% improvement in 42 days (p<0.02) and 49 days (p<0.10), respectively. A linear improvement in 1-42 days FCR by up to 3% was also observed as the level of butyrate increased in the starter feeds. Dietary supplementation of coated butyrate in starter feeds showed to have a lasting positive effect on broiler growth performance.
Fig. 2A Broiler chicks, 7 days, negative control Fig. 2B Broiler chicks, 7 days, 0.06% butyrate
Modulation of microbiota and impact on pathogens
Studies done by Galfi and coworkers (Galfi et al., 1991) have shown that butyrate increases the number of intestinal lactic acid and lactobacilli in butyrate-fed pigs, while decreasing the number of coliforms and E. coli. In his PhD-thesis Pérez Gutiérrez (2010) investigated microbial composition after including different additives in the feed of (weaning) pigs. The qPCR-data revealed an increase in lactobacilli in the butyrate group, while weaning piglets that received butyrate also had a more homogeneous microbial profile, which was regarded as a positive effect.
In studies of Van Immerseel and co-workers it was shown that butyrate, when present in the intestinal tract, was able to decrease Salmonella colonisation in broilers (Van Immerseel et al., 2005). The mode of action of this activity of butyrate seems at least partially mediated through modulation of gene expression. Butyrate specifically down-regulates Salmonella Pathogenicity Island 1 (SPI-1) gene expression, hereby preventing invasion of intestinal epithelial cells, one of the important steps of Salmonella pathogenesis in the bird (Gantois et al., 2006). The importance of an effective coating in order to get significant reduction in Salmonella colonisation in the ceca and internal organs in vivo was also shown in Fig 3. (Van Immerseel et al., 2005).
Fig. 3: Percentage of animals with a certain amount of Salmonella in caecum (n=25 per group).
A trial done at the University of Bologna (Bosi et al., 2010) was done to look at the possible impact of butyrate (coated and uncoated) on E. coli K88 (ETEC) infection in piglets. Fifty-four piglets (prone to ETEC intestinal adhesion) weaned at 21-28 days were used (three control groups of six animals, three challenged groups of 12 animals, balanced for litter and weight). Experimental diets were obtained with the addition of free or fat coated sodium butyrate. Full factorial design of two factors (four diets x challenge (E. coli K88) – yes/no), so groups were: 1) control diet, unchallenged; 2) 2 kg/T uncoated butyrate, unchallenged; 3) 2 kg/T coated butyrate, unchallenged; 4) control diet, challenged; 5) 2 kg/T uncoated butyrate, challenged; 6) 2 kg/T coated butyrate, challenged. The challenge with ETEC increased mortality in the control group (15% mortality) and had an impact on the growth rate of the piglets. The piglets receiving the uncoated sodium butyrate showed a lower mortality rate (5%) and were to a lesser extent influenced by growth than the challenged control group. In the group offered the coated sodium butyrate in the diet, none of the piglets died and the growth rate was only marginally influenced.
Butyrate – More than just an active compound
The effects on immune development, gut motility as well as the Salmonella effect via gene expression are only possible when the butyrate arrives in the lower parts of the intestinal tract. It is generally accepted that unprotected butyrate is quickly absorbed in the proximal part of the intestinal tract. Therefore, in order to get butyrate in significant levels available in the lower part of the intestinal tract the butyrate needs to be protected to achieve a target release.
In recent research done at the University of Illinois (Stein et al, 2012) the importance of a good coating was again demonstrated. The researchers investigated the disappearance kinetics of different sources of butyrate in diets fed to weanling pigs. Weanling pigs (n = 24; 8.0 ± 0.5 kg BW) were randomly allotted to 3 dietary treatments (6 replicate pigs per dietary treatment): 1) a control diet, 2) the control diet + 4kg/T of an uncoated butyrate (50% product, UCB), 3) the control diet + 4kg/T of a coated butyrate (30% product, CB). The dietary treatments were provided to pigs daily for 7 d as 3 times the estimated energy requirement for maintenance. On the last day of the experiment, all pigs were euthanized to collect samples of contents in the stomach, duodenum, jejunum, ileum, cecum, and proximal and distal colon. Concentrations of dry matter (DM) and butyrate were analyzed in all samples. There was a similar pattern of the concentration of butyrate in the digestive tract, indicating that the concentration of butyrate was greater in the stomach than in the duodenum and jejunum, and gradually increased in the ileum. Weanling pigs fed the CB diet had greater (P < 0.05) concentrations of butyrate in the jejunum (190 % μg butyrate/g digesta DM) and ileum (188% μg butyrate/g digesta DM) than weanling pigs fed the control diet (µg butyrate/g digesta DM put as 100%). In the cecum and colon, endogenous production was more variable among piglets and therefore no significant differences could be demonstrated. In conclusion, the coated butyrate in spite of lower percentage of butyrate (30%), increased significantly the concentrations of butyrate in the lower intestinal contents of pigs. Supplementation of the more concentrated (50%) but less protected butyrate did not result in significant differences in the amount of butyrate in the intestinal tract compared with the control group.
Fig. 4: Amount of butyrate in % compared to the unsupplemented control group.
Butyrate is a molecule of great interest to both human and veterinary medicine. Owing partly to the interest from human medicine, more and more profound knowledge on the mode of action has been obtained which allows for more sound advices to producers. In the current paper an attempt is made to summarize some of the newest developments on the research as well as to show experimental results in production animals; because, in the end the benefits to the efficiency of animal production are what matter. As shown above (Fig 4), butyrate has an effect on several levels (mucosal barrier, feed passage, microbiota, immune system, pathogens and others) and this combination of effects contribute to its general acceptance as helping for improved health as well as for improved performance.
References
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Gantois, I., R. Ducatelle, F. Pasmans, F. Haesebrouck, I. Hautefort, A. Thompson, J.C. Hinton, and F. Van Immerseel. 2006. Butyrate specifically down-regulates Salmonella pathogenicity island 1 gene expression. Appl Environ Microbiol. 72 (1): 946-949.
Guilloteau, P., R. Zabielski, J.C. David, J.W. Blum, J.A. Morisset, M. Biernat, J. Wolinski, D. Laubitz, and Y. Hamon. 2009. Sodium-butyrate as a growth promoter in milk replacer formula for young calves. J. Dairy Sci. 92: 1038-1049.
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Piva, A., M. Morlcchini, G. Casadie, P.P. Gatta, G. Biagi, and A. Prandini. 2002. Sodium butyrate improves growth performance of weaned piglets during the first period after weaning. Ital. J. Anim. Sci. 1: 35-41.
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Supplemental Sodium Butyrate Stimulates Different Gastric Cells in Weaned Pigs
Abstract
http://jn.nutrition.org/content/138/8/1426.full.pdf+html
Sodium butyrate (SB) is used as an acidifier in animal feed. We hypothesized that supplemental SB impacts gastric morphology and function, depending on the period of SB provision. The effect of SB on the oxyntic and pyloric mucosa was studied in 4 groups of 8 pigs, each supplemented with SB either during the suckling period (d 4–28 of age), after weaning (d 29 to 39–40 of age) or both, or never. We assessed the number of parietal cells immunostained for H+/K+-ATPase, gastric endocrine cells immunostained for chromogranin A and somatostatin (SST) in the oxyntic mucosa, and gastrin-secreting cells in the pyloric mucosa. Gastric muscularis and mucosa thickness were measured. Expressions of the H+/K+-ATPase and SST type 2 receptor (SSTR2) genes in the oxyntic mucosa and of the gastrin gene in the pyloric mucosa were evaluated by real-time RT-PCR. SB increased the number of parietal cells per gland regardless of the period of administration (P < 0.05). SB addition after, but not before, weaning increased the number of enteroendocrine and SST-positive cells (P < 0.01) and tended to increase gastrin mRNA (P = 0.09). There was an interaction between the 2 periods of SB treatment for the expression of H/K-ATPase and SSTR2 genes (P < 0.05). Butyrate intake after weaning increased gastric mucosa thickness (P < 0.05) but not muscularis. SB used orally at a low dose affected gastric morphology and function, presumably in relationship with its action on mucosal maturation and differentiation.
Introduction
Sodium butyrate (SB)9 is a very interesting molecule, because it is an energy source made available from bacterial fermentations, particularly for colonocytes. It also has important regulatory functions regarding cell proliferation, differentiation, and apoptosis, which differ between normal and cancer colorectal cells (1). Finally, SB modulates the gut microflora (2,3), depending on the adaptation of the bacteria to variations in chyme acidity (4).
The role of organic acids used as additives in the diet has received great attention. Acidification of infant formula with fermented products was proposed as a practical tool to prevent diarrhea (5). The protective action of organic acids can also be powerful when the passage from the maternal (milk) diet to the weaning diet is abrupt, as occurs in some farm animals. Butyrate is a digestion product normally released from milk triacylglycerol in the stomachs of suckled veal calves as a result of the sustained action of preintestinal lipases (6). In suckling pigs, the production of lactic acid from dietary lactose fermentation also contributes to acidifying the gastrointestinal contents (7), but it creates the conditions for delaying the full maturation of hydrochloric acid (HCl) secretion by the oxyntic mucosa (7). It can be hypothesized that dietary lactic acid or other organic acids can mimic the effects of the endogenous production of this acid. Immediately postweaning, supplementation of a diet with organic acids also reduced growth depression in piglets during this transition period (8). This effect could be related to the control of the development of Escherichia coli, as demonstrated with another organic acid salt, calcium formate (9). Among organic acids, the use of butyrate in the diet of weaning pigs has been less frequently studied. In piglets fed standard weaning diets, the amount of SB in the gastric content is very low (10) or not detected (11). Butyrate supplementation improved the gain:feed ratio in the first 2 wk postweaning (10). Conversely, SB administered for 6 wk postweaning did not affect growth performance (12).
The parietal cells in the oxyntic mucosa are responsible for HCl secretion through H+ production via the H+/K+-ATPase-dependent proton pump and Cl− secretion via an apical channel. The activation of acid secretion can act directly on the parietal cell (by the calcium-sensing receptor) (13) or indirectly via the gastric cell pathway. In this case, the release of gastrin from G cells in the pyloric mucosa acts on the endocrine enterochromaffin-like (ECL) cells of the gland. Following stimulation, ECL cells secrete histamine, causing the parietal cell to insert proton pumps (H+/K+-ATPase) into its apical membrane. Luminal acidification depresses gastrin secretion. Calcium formate added to the weaning diet for piglets reduces the number of HCl-secreting cells and H+/K+-ATPase gene expression in the oxyntic mucosa (14). Interestingly, direct inhibition of H+/K+-ATPase gene expression after diet acidification has also been observed in rainbow trout (15). Compared with formic acid, butyric acid is a weaker acid and it is possible that it does not inhibit gastric acid secretion.
In addition, it is not known if SB can stimulate a physiological pattern of gastric mucosa growth such as that observed for the colon (16) and cell proliferation from the jejunum to the distal colon (17). Furthermore, it has recently been shown that SB supplementation to formula-fed piglets from 3 to 10 d of age affected the development of the jejunal and the ileal mucosae (18). This raises the interesting point that butyrate supplementation in suckling pigs can hasten the maturation of the gut.
In this study, we hypothesize that supplemental SB alters the morphology and function of the gastric mucosa and that its action can be modulated by the period of administration. We tested this hypothesis by providing SB orally during the suckling and/or the postweaning periods in piglets and by assessing the effects of SB on various aspects of stomach morphology and physiology after the postweaning period.
Materials and Methods
Piglets, experimental design, and feeding.
For the ethical treatments of the piglets, the experimental procedures were carried out according to the guidelines of the French Ministry for Animal Research. The experiment involved 2 periods: 1) the suckling period from 4 d after birth until weaning at 28 d of age; and 2) the postweaning period from d 29 to 39–40 (day of slaughter). Two experimental factors were applied: butyrate before weaning (BE) and butyrate after weaning (AF). The combinations of these factors were defined as: CC, pigs never supplemented with SB; BC, pigs supplemented only before weaning; CB, pigs supplemented only after weaning; BB, pigs supplemented both before and after weaning.
For a total of 32 subjects, quadruplicates of Piétrain × Large White × Landrace piglets of the same birth weights and growth rates over the first 4 d of life were selected within litters and were assigned to the 4 dietary combinations. The experiment was conducted in 2 consecutive batches with 4 litters each. During the suckling period, an SB solution or saline solution used as the control treatment was delicately administered by esophageal tube twice daily (at 0900 and 1500) using a slightly curved brass tube (length, 12 cm; i.d., 3 mm; external diameter, 6 mm). The extremity ending with a bulbous part (length, 16 mm; wider external diameter, 12 mm) was made to avoid injury to the pigs. The other end was a luer-lock system that fit into 5–10 mL plastic syringes. SB was supplied at a concentration of 0.3% of the daily dry matter (DM) intake. Milk intake for each piglet was estimated during the suckling period from its growth rate using the equation of Noblet and Etienne (19).
For the postweaning period, the amount of feed was increased from 10 to 80% of the Institut National de la Recherche Agronomique recommendation (20) between d 28 and 32 to reduce the incidence of indigestion that can occur with immediate ad libitum consumption. This amounted to 70 g/kg body weight0.75 at d 32. Amounts of feed covering 100% of the requirements were offered from d 33 to the end of the experiment. The weaning diets were offered twice daily at 0900 and 1500. Piglets consumed water ad libitum.
During the suckling period, the piglets stayed with their mother in the farrowing unit. At d 28, they were transported to the weaning building. There they were housed in individual cages with a mesh floor in a temperature-controlled room.
Treatments and diets.
Two solutions were prepared for oral administration during the suckling period: 1) saline solution (9 g NaCl / L) for the control groups (CC and CB); and 2) SB solution (60 g/L, Sigma, 303410) dissolved in saline solution for the SB groups (BC and BB). The pH was adjusted to 7.0 by adding NaOH. Two weaning diets offered only from the day of weaning were formulated (Tables 1 and 2). For the CB and BB groups, SB was introduced (3 g/kg) in 1 of these starter diets by replacing corn starch. Thus, the daily consumption of SB ranged from 0.4 to 0.7 g/d during the suckling period and 0.7 to 1.5 g/d in the postweaning period. No antimicrobial agent was added to the diets. These diets were offered to piglets as a mash with a feed:water ratio of 1:1 by weight during the first 3 d after weaning and 2:1 thereafter.
View this table:
TABLE 1
Control diet composition on an as-fed basis1
View this table:
TABLE 2
Effect of butyrate supplementation before or after weaning, at both times, or never on gastric morphological parameters of pigs1
Pig slaughter and tissue sampling.
The piglets from 2 litters were randomly killed 4 h after the last meal on d 11 and those from the remaining 2 litters on d 12 after weaning (d 39–40 of age). The piglets were stunned by electric shock and then exsanguinated. For each pig, a midline abdominal incision was made and the whole gastrointestinal tract was gently removed. The stomach was separated, opened along the greater curvature, emptied of its contents, and rinsed with twice-distilled water. Whole thickness tissue specimens of ∼1 cm2 were removed from the oxyntic and pyloric gland areas near the greater curvature and from the antral region. Tissue samples were pinned tightly to balsa wood and were fixed in 10% buffered formaline (immunohistochemistry) or Bouin’s solution (morphometry) for 24 h. The tissue samples were then removed from the fixative and washed in 5.14 mol/L ethanol. The specimens were then dehydrated in a graded series of ethanol and embedded in paraffin. For each pig, additional samples of the entire oxyntic and pyloric walls were collected for molecular biology, snap-frozen in liquid nitrogen, and stored at −80°C until analysis.
The pH of the fresh gastric digesta was determined immediately upon collection using a pHmeter (704 model, Metrohm) and the content was then frozen, freeze-dried, milled, and stored for analysis.
Muscularis and mucosa morphometry.
Tissue sections of 5 μm thickness were cut from paraffin blocks to estimate the muscularis and mucosa thickness. The tissue sections were then stained with hematoxylin and eosin according to a standard protocol. Thirty whole cross-sections of gastric muscularis and mucosa per slide were measured using Axio Vision 4.3 software (Zeiss) under a light microscope (Zeiss).
Immunohistochemistry.
Adjacent formalin-fixed sections (5 μm) underwent immunohistochemical staining for detecting parietal and endocrine cells. We used microwave treatment for unmasking relevant antigenic sites before immunodetection. All the antibodies used in this study are listed in Supplemental Table 1.
Immunostaining of parietal cells was performed as previously reported (14). Briefly, the sections were treated with 90 mmol/L H2O2 in methanol for 30 min to block endogenous peroxidase activity, then with normal goat serum for 1 h, followed by a primary antibody against the α-subunit H+/K+-ATPase incubated at 4°C overnight by a biotin-conjugated goat anti mouse IgG and then by ABC complex (Vector Laboratories). The immune reactions were visualized applying a 3–3′-diaminobenzidine chromogen solution (Vector Laboratories).
For each pig, we counted all the parietal cells in 20 randomly selected glands located perpendicularly to the surface of the mucosa using an optical microscope. The depth of the lamina propria, from the pits to the muscularis mucosae, was measured in the same areas using a Zeiss Axioplan microscope (10× objective) connected to KS 300 image analysis software (Kontron Elektronic).
Immunostaining of the endocrine cells was performed using the indirect double-labeling immunofluorescent technique. Anti-chromogranin A antibody, which labels endocrine cells (21,22), was used in association with anti-somatostatin (SST) antibody for the oxyntic mucosa and anti-gastrin antibody was used for the pyloric mucosa. The sections were incubated at 4°C overnight in a solution containing chromogranin A/SST or SST/gastrin mixtures of the primary antibodies. After washing in PBS, the sections were incubated in the dark for 1 h with a mixture of goat anti-rabbit antibody labeled with fluorescein isothiocyanate and goat anti-mouse antibody labeled with Alexa 594 (Supplemental Table 1).
Negative controls to prove the specificity of the secondary antibodies were obtained by incubating the sections without the primary antibody or with appropriate nonimmune γ globulins.
For each pig, all the endocrine cells in 20 randomly selected glands located perpendicularly to the mucosal surface were counted using a Zeiss Axioplan microscope equipped with the appropriate filter cubes for discriminating between fluorescein isothiocyanate and Alexa 594 stainings.
Gene quantification by real-time RT-PCR.
To quantify mRNA abundance for ATPase, SST type 2 receptor (SSTR)-2 and gastrin genes in the fundic and pyloric mucosa, total RNA was isolated and its integrity controlled as previously reported (9).
For each gene investigated, 2 pairs of primers were designed on the specific pig nucleic acid sequence (GenBank) by Primer 3 (23) (Supplemental Table 2). An absolute quantitative analysis, using an external standard curve for each gene, was performed in a LightCycler instrument (Roche), as described previously (14). Data were expressed as gene copies per microgram RNA.
Statistics.
ANOVA using the GLM procedure of SAS (version 8.1, SAS Institute) with a 2-level full factorial design was carried out: BE, AF, and interaction. Batch and litter within batch were also included in the model. When the P-value for the BE by AF interaction was <0.10, the following preplanned orthogonal contrasts were performed: “Butyrate whichever period vs. never butyrate,” (CB+BC+BB) vs. CC; “Butyrate, 1 period vs. 2 periods,” (CB+BC) vs. BB; “Butyrate, only before vs. only after,” BC vs. CB. The values presented are least square means ± SEM and effects were considered significant at P < 0.05. The values for gene expressions did not display a normal residue distribution. Therefore, a log base 10 transformation of the data were used. The correlation between H/K-ATPase and SST type 2 receptor (SSTR2) genes expressions was also calculated.
Results
Except for 2 piglets, the piglets from the experimental groups remained clinically healthy during the study. One pig from the BB group of the first batch and 1 pig from the BC group of the 2nd batch had to be removed from the experiment, because they did not consume the weaning feed. Excluding those subjects, the final body weights were 10.5, 11.4, 11.0, and 12.1 ± 0.4 kg, respectively, for CC, BC, CB, and BB groups (effect of SB supplementation before weaning, P < 0.05). SB did not change growth before weaning and the pooled daily gain from d 4 to 28 was 284 ± 17 g. In the postweaning period, the interaction between SB supplementation before and after weaning tended to be significant (P = 0.10). The daily gains of the BC (195 g), CB (174 g) and BB (192 g) groups were higher than that of the CC group (133 g, pooled SE=12 g) (P < 0.05).
The weight of the empty stomach relative to body weight was 9.68 ± 0.35 g/kg and the pH of the gastric contents was 3.40 ± 0.09 and did not differ among the groups. The amount of residual DM in the stomach, calculated as a percentage of the DM intake of the last meal and normalized for body weight, was greater (P < 0.05) in the groups that received SB during the suckling period (BC, 45.0; BB, 51.4) than in those that did not (CC, 36.9; CB, 42.1 ± 3.8; pooled SE = 3.8 gastric residual DM amount/DM intake, %). SB addition postweaning and the interaction between the 2 SB supplementation periods did not affect this variable.
The depth of the oxyntic gland tended to be increased by butyrate BE (P = 0.108) (Table 2). The interaction between SB addition before and after weaning approached significance for the number of parietal cells per gland (P = 0.097), which was increased by the administration of SB whatever the period of treatment (see also Fig. 1A, CC group; Fig. 1B, BB group). The number of parietal cells per 100-μm depth of the gland tended (P = 0.099) to be increased with the addition of SB after weaning. The number of enteroendocrine cells, per gland and per 100-μm depth of the gland, was strongly increased by SB after weaning (Fig. 1C, CC diet; Fig. 1D, BB diet). SB after weaning also increased the number of SST cells (Fig. 1E, CC diet; Fig. 1F, BB diet). However, when these cell numbers were expressed per gland, SB addition during the suckling phase tended to have an effect (P = 0.082). All the SST-positive cells were also chromogranin A-positive. The 2 SB treatments interacted to affect the ratio of SST-positive:total enteroendocrine cells. The pigs receiving SB in both periods had relatively more SST-positive cells than the pigs receiving SB only before or after weaning. Finally, the counts of gastrin-positive cells in the pyloric mucosa was not affected by the treatments.
FIGURE 1
Effect of the diet on parietal cells in unsupplemented pigs or those supplemented with butyrate before and after weaning. The oxyntic mucosa was immunostained with an antibody to the α-subunit of H+/K+-ATPase (A,D) or double immunostained with antibodies to chromogranin A (B,E) and SST (C,F). Antibody binding was detected by the ABC method (A,D) and by immunofluorescent methods (B,C,E,F). Parietal cells were more numerous in the BB group (D) than in the CC group (A). The cells coexpressing chromogranin A (B,E) and SST (C,F) immunoreactivity were spread throughout the gland (arrows); BB pigs showed an elevated number of chromogranin A positive cells (E) and SST-positive cells (F) compared with CC pigs (B and C, respectively).
Butyrate supplementation after weaning increased gastric mucosa thickness, with no effect on the muscularis thickness. Mucosal thickness in the CB (467 μm) and BB (482 μm) groups were greater than in the CC (403 μm) and BC (431 μm; pooled SE = 31 μm) groups. SB treatment in the suckling period did not affect this variable.
There was an interaction between the pre- and postweaning periods of SB supplementation for H/K-ATPase and SSTR2 mRNA abundance (Table 3) but no significant contrast for H/K-ATPase. Supplying SB in either period only tended to increase SSTR2 (P = 0.098) mRNA abundance compared with SB addition in both periods. In the pyloric mucosa, feeding SB postweaning tended to increase gastrin mRNA (P = 0.087).
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TABLE 3
Effect of butyrate supplementation before or after weaning, at both times, or never on gene expression in the stomach mucosa of pigs1
Discussion
The major findings of the present study are that SB supplementation postweaning increased the densities and numbers of enteroendocrine and SST cells in the fundic mucosa of pigs.
Enteroendocrine cells in the oxyntic mucosa include various endocrine/paracrine cells (ECL cells, A-like cells secreting ghrelin and obestatin, and D cells secreting SST). For the intestine, the enteroendocrine L cells, which produce the intestinotrophic glucagon-like peptide-2, have been proposed to be target cells for butyrate (16). The selective induction of enteroendocrine cells in the stomach by SB has, to our knowledge, never been reported before. It is generally assumed that oral SB, at a low gastric pH, is rapidly protonated and absorbed by nonionic diffusion in the stomach (24), but the moderate presence of the monocarboxylate transporter 1 in the basolateral surface of gastric epithelial cells could facilitate butyrate exchanges (25). The movements of butyrate can also be favored by the presence of the monocarboxylate transporter 2 in the area of parietal cells (25). However, there is no evidence yet that enteroendocrine cells selectively utilize butyrate.
The increased numbers of SST-positive enteroendocrine D cells after SB addition postweaning can be explained by the action of this peptide as an inhibitory feedback messenger on acid secretion, which has already been observed in piglets fed free calcium formate (14). This apparently contrasts with the trend of increased gastrin mRNA abundance. Indeed, SST has an inhibitory effect on gastrin gene transcription (26). But, if the number of SST-positive cells is related to the total number of enteroendocrine cells, the effect of SB is more complex and variable according to the period of supplementation. Pigs that received SB for both periods had a relatively greater number of SST cells than pigs that were supplemented with SB only before or after weaning. Conversely, in the BB group, a negative trend was seen for mRNA abundance of SSTR2, which mediates the action of SST in parietal, ECL, and G cells. Both observations could indicate a sort of long-term adaptation to SB supplementation. The pattern of expression of SSTR2 mRNA abundance probably reflects the various cell populations on which the receptors are found. Direct feedback after gastrin secretion should be excluded; in fact, SSTR2 gene expression was not reduced in gastrin-knockout mice or gastrin/CCK-knockout mice (27). Finally, we should also consider a direct effect of SB on the growth or activity of D cells, because SB induces SST production on cultivated cells (28).
The control of luminal pH in the gut is critical to the digestive function and the integrity of the different parts of the gastrointestinal tract. This is achieved using various mechanisms mainly governed by acid-sensing primary afferent neurons (29), resulting in feedback on gastric acid secretion, mucosal function, and motility. Because SB was used here at a low dose and is a moderate acid, the lack of reduction in gastric pH with SB treatment is not surprising and agrees with other observations that were reached using the same dose of SB (10). Calcium formate (12 g/kg feed) decreased parietal cell numbers and H+/K+-ATPase gene expression in the porcine oxyntic mucosa (14). These signs of short- or long-term adjustments were not observed with SB at the present dose.
One mechanism used by the digestive system to control luminal pH is the delay of gastric emptying. Manzanilla et al. (10) have shown that SB after weaning did not affect feed intake but increased the DM percentage of the gastric contents, which is an indicator of slower gastric emptying or of faster liquid phase outflow from the stomach. In the present study, residual DM in the stomach at slaughter increased in pigs fed SB before weaning, whatever the treatment after weaning (30). However, this was accompanied by increased feed intake. The persistence of an effect after the suspension of the supplementation with SB suggests that control of the pH in the digesta contents flowing to the duodenum is not sufficient to explain our observation. This is additionally supported by the fact that there was no effect on residual DM with the SB treatment after weaning (30). Mechanisms involving the long-term effects of butyrate could be advocated. We herein present data showing that some morphological variations (parietal cells, SST+ cells) in the fundic mucosa tend to remain for at least 1 wk after the suspension of SB treatment. More data, also including information from physiological events in the intestine, are required to better understand gastric physiology mechanisms.
The number of parietal cells per gland in the oxyntic mucosa increased in the pool of SB treatments compared with the controls. Acid secretion is a high energy-demanding process and SB can rapidly penetrate through cell membranes. Butyrate supported H+ secretion, although less effectively than glucose, acetate, and pyruvate in the isolated gastric mucosa of neonatal pigs (31). However, there is no indication that SB can stimulate the proliferation of parietal cells, notwithstanding the demonstrated effect on colonocytes (16). Here, gastrin gene expression also tended to increase after SB supply. This observation may be related to the delay of gastric emptying, because stomach distension is known to stimulate gastric acid production. Recent research demonstrates that gastrin, besides its effect on gastric secretion, regulates the organization of the gastric mucosa (32) and also acts as a proliferation agent on parietal cells and ECL cell progenitors (33,34). In our trial, SB provided postweaning also tended to increase the number of parietal cells per unit depth of the gland but did not affect the depth of the gland. This result is consistent with the observations of Kotunia et al. (18) who did not find any effect on the plasma gastrin level in neonatal pigs fed SB in milk formula, but contrasts with those of Bakke et al. (35) who reported that SB increased the number of parietal cells in parallel with the total number of mucosal cells when hypergastrinaemia was induced in rats. However, a paracrine effect of gastrin without any change in its blood concentration, via activation of other growth factors (36), can be hypothesized.
SB did not affect the number of gastrin-secreting cells in our trial. In the piglets provided with oral SB, the effect should have persisted for 11 – 12 d after the end of the treatment. The lifetime of parietal cells in mice is estimated to be ∼80 d (37). Therefore, it can be hypothesized that the persistent effect of SB on parietal cell numbers is the residual result of the same effect seen on parietal cells with SB supplementation postweaning. However, the number of parietal cells per unit depth of the gland was not changed by the preweaning provision of SB. Indeed, the depth of the gland tended to increase after preweaning SB administration; thus, the increase of the cell number reflected, at least partially, an overall effect of SB on oxyntic gland development.
Notwithstanding the average increase in the number of parietal cells with oral SB and the trend for increased gastrin mRNA abundance with SB after weaning, H+/K+-ATPase gene expression in the oxyntic mucosa was not changed by the supplementation. In a previous experiment supplementing piglets with calcium formate, individual variations in ATPase gene expression were partially explained by individual variations in parietal cell numbers (14). However, the time elapsed between the last meal and the killing differed between experiments (2 h vs. 4 h here). To the best of our knowledge, the synchrony between the expressions of genes related to the short-term control of gastric secretion has not been studied. More information about the effect of the time from the stimulus induced by the meal and/or the presence of feed in the stomach on gene expression would be useful for a better interpretation of such experiments.
The data on different gastric cells can be integrated with the results from mucosa morphometry obtained in the antrum region. SB treatment after weaning resulted in increased mucosal thickness. This observation suggests that SB action on stomach tissue is relevant to its functional development. Elevated growth of the gastric mucosa may result from high proliferation and/or low apoptosis rates. Kien et al. (17) showed that cecal butyrate infusion at a rate equal to that produced in the colon did not affect the apoptosis index but caused a 78–119% increase in cell proliferation in the jejunum, ileum, distal colon, and cecum. Such information is still not available for the stomach.
In conclusion, our investigation provides evidence that SB has a complex impact on porcine gastric morphology and function that cannot be explained by its acid function. Supplementation with butyrate from weaning to death stimulated more cells to differentiate into enteroendocrine cells; furthermore, dietary butyrate increased the number of parietal cells per gland, whatever the period of supplementation (including a trend for pigs after 11–12 d of suspension of the treatment). This is the first study to our knowledge that demonstrates the ability of supplemental SB to affect cellular mechanisms of differentiation and to control tissue growth in the normalm healthy stomach, such as is observed in the large intestine in vivo or in in vitro systems. Finally, oral SB proved to be of interest for stimulating growth performance and feed intake when provided to young pigs, especially before weaning.