ABSTRACT
Two experiments (E) were carried out to evaluate the effects of fumaric acid and an acidifier blend [composed by calcium formate, calcium lactate and medium-chain fatty acids (capric and caprylic)] in piglet diets containing colistin (40 ppm) or halquinol (120 ppm) on performance, diarrhea incidence (E1), organs relative weight, pH values, intestinal morphometry and microbiota (E2). In E1, 192 and E2, 24 piglets weaned at 21-day-old were randomly assigned to blocks with 2x2 factorial arrangement of treatments [absence or presence of fumaric acid x absence or presence of acidifier blend], six replicates of eight (E1) and one piglet per pen (E2). For E1, the treatments were control (CD): no acidifier product + 40 ppm of colistin, FA: fumaric acid in absence of acidifier blend, AB: acidifier blend in absence of fumaric acid and, AF+AB: presence of fumaric acid and acidifier blend. For E2, the pre-starter I diet were used and the same treatments as E1 evaluated. No treatment effects (P>0.05) were observed on performance, diarrhea incidence (E1), gut pH values and duodenum morphometry of piglets (E2). However, the addition of AB increased (P<0.05) large intestine relative weight and, FA addition decreased (P<0.05) pancreas relative weight, jejunum villi height and, total coliform and E. coli counts in cecum. The inclusion of FA and AB in diets containing colistin or halquinol did not improve performance, although FA exerted an inhibitory effect on cecum microbiota.
Key words:
acidifiers; diarrhea; nutrition; pig; weaning
INTRODUCTION
Early-weaning is one of the most critical management practices in piglet’s life, which constitutes a challenge for them and results in poor growth performance, reduced immunity, and an imbalanced microbiota. The main factors contributing to these results are low feed intake and onset of diarrhea (Barnett et al. 1989BARNETT KL, KORNEGAY ET, RISLEY CR, LINDEMANN MD AND SCHURIG GG. 1989. Characterization of creep feed composition and its subsequent effects on immune response, scouring index and performance of weanling pigs. J Anim Sci 67(10): 2698-2708. ). At weaning, the piglets that were physiologically adapted to digest the sow’s milk are abruptly submitted to a diet with high amount of vegetable ingredients, usually a cereal like corn as main energy source and high in starch (Vente-Spreeuwenberg et al. 2003), and another vegetable protein source like soybean meal, for which they are not prepared to digest due to its insufficient production of gastric hydrochloric acid that leads to an increased pH and decreased activity of proteolytic enzymes. Besides that, the lactic acid produced by fermentation of lactose is primarily responsible for acidification of suckling pig stomach and, according to Barrow et al. (1977BARROW PA, FULLER R AND NEWPORT MJ. 1977. Changes in the microflora and physiology of the anterior intestinal tract of pig weaned at 2 days, with special reference to the pathogenesis of diarrhea. Infect Immun 18: 586-595. ), this acid acts as HCl-production inhibitor. Also, the presence of large amount of soybean meal in piglets’ diet might cause hypersensitivity reactions which result on altered bowel structures (the villi) hindering the digestion of feed and absorption of nutrients (Li et al. 1990LI DF, NELSSEN JL, REDDY PG, BLECHA F, HANCOCK JD, ALLEE GL, GOODBAND RD AND KLEMM RD. 1990. Transient hypersensivity to soybean meal in the early-weaned pig. J Anim Sci 68(6): 1790-1799.). Therefore, the use of enhancing additives can be an alternative to compensate these deficiencies.
Concerning the appearance of resistant bacteria and residues in animal products, the European Union has been prohibited the use of antibiotics as performance-enhancing antimicrobial compounds. Thus, the interest for alternative products, such as organic acids and their salts have increased by the consumer, due to its antimicrobial activity and safety (Blank et al. 2001BLANK R, SAUER WC, MOSENTHIN R, ZENTEK J, HUANG S AND ROTH S. 2001. Effect of fumaric acid supplementation and dietary buffering capacity on the concentration of microbial metabolites in ileal digesta of young pigs. Can J Anim Sci 81(3): 345-353. , Costa et al. 2011COSTA LB, ALMEIDA VV, BERENCHTEIN B, TSE MLP, ANDRADE C AND MIYADA VS. 2011. Aditivos fitogênicos e butirato de sódio como alternativas aos antibióticos para leitões desmamados. Arch Zootec 60(231): 733-744. ). According to Cherrington et al. (1991CHERRINGTON CA, HINTON M, MEAD GC AND CHOPRA I. 1991. Organic acids: chemistry, antibacterial activity and pratical applications. Adv Microb Physiol 32: 87-108.), these products leave no residues in carcass and do not promote emergence of resistant bacteria, being a natural, safe, and inexpensive alternative to antibiotics in order to improve the health and performance of animals. For pigs, the most commonly used acids are acetic, formic, propionic, lactic, citric and fumaric acid (Partanen 2002PARTANEN K. 2002. Using organic acids in pig feeding as an alternative to antibiotics feed additives. In: Simpósio sobre Manejo e Nutrição de Aves e Suínos e Tecnologia na Produção de Rações. Proceedings... Campinas: Colégio Brasileiro de Nutrição Animal, p. 45-62.), and the efficiency of organic acids in improving piglet’s growth performance have been confirmed by other studies (Giesting and Easter 1985GIESTING DW AND EASTER RA. 1985. Response of starter pigs to supplementation of corn-soybean meal diets with organic acids. J Anim Sci 60(5): 1288-1294. , Radecki et al. 1988RADECKI SV, JUHL MR AND MILLER ER. 1988. Fumaric and citric acids feed additives in starter pig diets: effect on performance and nutrient balance. J Anim Sci 66(10): 2598-2605., Radcliffe et al. 1998RADCLIFFE JS, ZHANG Z AND KORNEGAY ET. 1998. The effects of microbial phytase, citric acid, and their interaction in a corn-soybean meal-based diet for weanling pigs. J Anim Sci 76(7): 1880-1886. ).
Therefore, this study aimed to evaluate the effects of dietary supplementation of fumaric acid and an acidifier blend [calcium formate, calcium lactate and medium-chain fatty acids (capric and caprylic)] on growth performance, organs weight, pH, morphometry and intestinal microbiota of weaned piglets.
MATERIALS AND METHODS
All experimental procedures were previously approved by the Animal Ethics Commitee of the Veterinary and Animal Science College from São Paulo State University (UNESP), under protocol number 187/2012-CEUA and, in accordance with directive 2010/63/EU.
EXPERIMENT 1
The experiment was conducted in a nursery facility at a commercial farm in city of Patos de Minas, Minas Gerais State, Brazil. The nursery stall was divided into three rooms, with ceiling height of 2.40 m, and eight pens each, totaling twenty-four pens. Each pen had 4.64 m2 with a partially slatted and not suspended flooring, two nipple-type drinkers and a semi-automatic feeder for seven piglets. A hundred forty and four male piglets (castrated) and forty-eight females, totaling 192 animals, from Agroceres PIC genetics (Pietrain/Landrace x Duroc/Landrace) were weaned at 21-day-old with average initial body weight of 6.80 ± 0.89 kg. The control temperature within each nursery room was carried out with the management of lateral curtains and heaters.
The experiment duration lasted 41 days and the piglets were fed three types of diets (Table I) according to a nursery feeding system in phases: pre-starter diet I (PI) in the first 14 days after weaning (from 21- to 35-day-old), pre-starter diet II (PII) in the following 14 days (from 36- to 49-day-old) and, starter diet (SI) until the end of the experiment (from 50- to 62-day-old). The formulated diets followed the nutritional requirements proposed by Rostagno et al. (2011ROSTAGNO HS, ALBINO LFT, DONZELE JL, GOMES PC, OLIVEIRA RF, LOPES DC, FERREIRA AS, BARRETO SLT AND EUCLIDES RF. 2011. Composição de alimentos e exigências nutricionais de aves e suínos (tabelas brasileiras), 3ª ed., Viçosa: Universidade Federal de Viçosa, 252 p.), except for calcium. All animals hadad libitum access to feed and water.
The experimental design was a randomized block design with four treatments, six replicates and eight animals per experimental unit, in a factorial arrangement [absence or presence of fumaric acid (FA) x absence or presence of acidifier blend (AB)]. The variables considered for blocks formation were the weight and sex of animals.
The treatments were CD: PI, PII and SI diets without acidifier product; FA: PI, PII, and SI diets containing 0.8%, 0.4% and 0.2% of fumaric acid, respectively; AB: PI, PII, and SI diets containing 0.3%, 0.3% and 0.2% of acidifier blend, respectively; AB + FA: PI, PII, and SI diets containing 0.8%, 0.4% and 0.2% of fumaric acid and 0.3%, 0.3% and 0.2% of acidifier blend respectively. For all treatments, 40 ppm of colistin were added to PI and PII diets and, 120 ppm of halquinol were added to SI diets. The acidifier product used was a mixture of calcium formate, calcium lactate salts and medium-chain fatty acids (capric and caprylic). The inclusions of acidifier, the colistin sulphate and the halquinol in feed were possible by the substitution of equivalent amount of inert material (kaolin).
At beginning of experiment and at each diet switch, the piglet weight was measured individually. At periods 0 to 14, 0 to 28 and 0 to 42 days after weaning, the daily feed intake, daily weight gain and feed:gain ratio were evaluated. During the first 14 days of experiment, the diarrhea incidence was evaluated twice a day by the same observer. Diarrhea status was considered when visually faeces had fluid consistency. The values of diarrhea incidence were calculated as mean percentage of animals with diarrhea in the pen (p), and the data were subjected to angular transformation: p’ = arcsine*[√(p/100)] for further analysis of variance. The animals were housed in pens that have not undergone to previous disinfection. The diet pH was evaluated according to the following methodology (AOAC 1990): 5 g of feed sample was weighed into a 100 mL becker, following 25 mL of de-ionized water addition. After mixing the solution and waiting for 30 min, the pH of solution was registered by a portable Tecnopon pH meter (model mPa-210P, Piracicaba, Brazil).
All data were analyzed using the GLM procedure of SAS 9.0 (SAS 2002). Tukey’s multiple range tests were applied to test significant differences between treatments. For all of statistical analysis for this work, the pen served as an experimental unit. Variability in the data was expressed as coefficient of variation (CV) and a probability level of (P<0.05) was considered as statistically significant.
EXPERIMENT 2
The second experiment was conducted in experimental swine production facilities from Faculty of Veterinary Medicine and Animal Science at São Paulo State University (UNESP), Botucatu Campus, Brazil with twelve castrated males and twelve females piglets from commercial genetics (Landrace x Large White) weaned at 21-day-old with an average body weight of 6.46 ± 0.24 kg. The nursery facility had metal suspended pens of 1.75 m2 and, a partially slatted flooring with a compact concrete floor under the heater, being equipped with one feeder, one nipple-type drinker and one heater.
The experimental design was a randomized block design with four treatments, six replicates and one animal per experimental unit. The experiment duration lasted 14 days, during which the animals were fed pre-starter I diets (PI) (Table I). All treatments were the same as defined in Experiment I.
At 14th experimental day, all piglets were slaughtered after electrical stunning. A longitudinal incision was made along abdominal cavity for removal of digestive organs. The gastrointestinal tract (with contents) was weighed by full and after removing the stomach, small intestine, cecum and large intestine content, they were weighed again, one by one, to determine its absolute weight. The liver, pancreas, spleen and kidneys were also weighed in order to determine their absolute weights. After this procedure, the relative weight of each organ was calculated based on the empty carcass weight (weight with no digesta content). Also pH readings of stomach, jejunum, ileum and colon contents were measured immediately after slaughter by a portable Tecnopon pH meter (model mPa-210P, Piracicaba, Brazil).
The cecum of each animal was removed with its content and stored in styrofoam boxes with ice for further laboratory analysis. The total coliform and Escherichia coli counts were measured from 10 g of cecal content, and then added to 90-mL of 0.85% saline and homogenized for 2 min, obtaining a 10:1 dilution. After, a serial decimal dilutions were prepared, transferred to 1-mL of ECTM Petri dishes and incubated at 35°C for 24-hour (AOAC 2000). The Lactobacillus spp. count was performed from decimal serial dilutions as described for Escherichia coli count, transferring 0.1-mL into Petri dishes containing Man-Rogosa-Sharpe (surface sowing), incubated at 30°C for 5 days in a drying air oven with 5% CO2 atmosphere (Downes and Ito 2001DOWNES FP AND ITO K. 2001. Compendium of methods for the microbiological examination of foods, 4th ed., American Public Health Association: Washington, USA, 676 p.). Microflora enumerations were expressed as log10colony-forming unit (cfu) per gram.
For gut morphometry analysis, samples were collected from 1.5 cm of duodenum and jejunum initial and middle portions which were immersed in 10% formalin solution for 48-hour. Then, samples were immersed in 70% etanol solution, dehydrated in ethyl alcohol (increasing concentrations). The tissue fragments were cut about 1.0 cm in each segment, diaphanized in benzene and embedded in paraffin. Four semi-serial sections of segments (jejunum and duodenum) with 5 μm thickness were performed for each animal. After processing, the sections were placed on slides, stained by hematoxylin and eosin technique and examined under a light microscope for villi height and width, crypt depth measurements using Leica QWin image processing and analysis workstation software. Thirty readings were performed for each animal and each segment. The final result was the average of 30 measurements.
The scanning electron microscopy technique was used to evaluate the villi density. Duodenum and jejunum samples collected after slaughter were fixed in 3% glutaraldehyde, washed in 0.1 M sodium cacodylate, buffered in pH 7.2, post-fixed in 1% osmium tetroxide. Then, samples were washed in the same buffer and dehydrated in an increasing ethanol concentration. The tissue samples were soacked into isoamyl acetate solution for 3-hour. Drying was performed obtaining the critical point of liquid CO2 in a dry air oven SEM model 850. A Denton Desk II model vacuum device was used for sputter coated (gold-palladium). The samples were mounted over supports of 1.0 cm in diameter and thickness, using metal tape, observed and registered by a scanning electron microscope JEOL JSM 5410, operated at accelerating voltage of 15 kV. The eletron micrographs from four samples were taken counting the villi. Then, to assess the approximate magnitude of the absorptive area per 1 mm2of the intestinal mucosa (mm2/mm2), the surface of villi was calculated and multiplied by the number of villi, using the following equation: πr2+ 2πrh, where r is the average radius (villus width/2) and h is the villi height (Skrzypek et al. 2010SKRZYPEK T, VALVERDE PIEDRA JL, SKRZYPEK H, KAZIMIERCZAK W, SZYMANCZYK SE AND ZABIELSKI R. 2010. Changes in pig small intestinal absorptive area during the first 14 days of life. Livest Sci 133: 53-56. ).
All data were analyzed using the GLM procedure of SAS 9.0 (SAS 2002). Tukey’s multiple range tests were applied to test significant differences between treatments. Variability in the data was expressed as coefficient of variation (CV) and a probability level of (P<0.05) was considered as statistically significant.
RESULTS AND DISCUSSION
EXPERIMENT 1
The use of AB decreased (P<0.05) pH values of diets. This reduction was increasingly higher for the acidifier blend, fumaric acid and fumaric acid in combination with the mixture of acids (Table II). No interactions and no effect of main factors (P>0.05) were registered on daily feed intake (DFI), daily weight gain (ADG), feed:gain ratio (F:G) and diarrhea incidence (DI) of piglets (Table III) on studied periods. These results corroborate those reported by Gomes et al. (2011GOMES FE, FONTES DO, VASCONCELLOS CHF AND SILVA FCO. 2011. Ácido fumárico e sua combinação com ácido lático ou propionato de cálcio em dietas de leitões recém-desmamados. Arq Bras Med Vet Zootec 63(3): 678-686. ), that the addition of 0.5% of fumaric acid and its combination with 0.5 % lactic acid and 0.5% calcium propionate did not improve growth performance of piglets weaned at 15-day-old. Similarly, Zentek et al. (2013ZENTEK J, FERRARA F, PIEPER R, TEDIN L, MEYER W AND VAHJEN W. 2013. Effects of dietary combinations of organic acids and medium chain fatty acids on the gastrointestinal microbial ecology and bacterial metabolites in the digestive tract of weaning piglets. J Anim Sci 91(7): 3200-3210. ) found no effect on growth of weanling piglets fed diets containing the mixture of medium-chain fatty acids (capric and caprylic).
The addition of organic acids and their salts in diet of weanling pigs, such as fumaric acid (Giesting and Easter 1991GIESTING DW AND EASTER RA. 1991. Effect of protein source and fumaric acid supplementation on apparent ileal digestibility of nutrients by young pigs. J Anim Sci 69(6): 2497-2503.), lactic acid (Tsiloyiannis et al. 2001TSILOYIANNIS VK, KYRIAKIS SC, VLEMMAS J AND SARRIS K. 2001. The effect of organic acids on the control of porcine post-weaning diarrhoea. Res Vet Sci 70(3): 287-293. , Silva 2002SILVA MC. 2002. Efeito da adição de acidificantes e suas combinações na alimentação de leitões desmamados sobre o desempenho. In: Reunião Anual da Sociedade Brasileira de Zootecnia, Recife. Proceedings ... Campinas: ABZ. ), calcium formate (Partanen and Mroz 1999PARTANEN KH AND MROZ Z. 1999. Organic acids for performance enhancement in pig diets. Nutr Res Rev 12(1): 117-145. , Bosi et al. 2005BOSI P, SARLI G, CASINI L, DE FILIPPI S, TREVISI P, MAZZONI M AND MERIALDI G. 2005. Effect of dietary addition of free or fat-protected calcium formate on growth, intestinal morphology and health of Escherichia coli K88 challenged weaning pigs. Ital J Anim Sci 4(2): 452-454. ) or the combination as lactic acid, formic acid and phosphoric acid (Freitas et al. 2006FREITAS LS, LOPES DC, FREITAS AF, CARNEIRO JC, CORASSA A, PENA SM AND COSTA LF. 2006. Avaliação de ácidos orgânicos em dietas para leitões de 21 a 49 dias de idade. Rev Bras Zootec 35(4): 1711-1719. ) have resulted in benefits on growth performance, while other researches have shown negative responses on piglets’ growth performance due to formic acid usage (Manzanilla et al. 2004MANZANILLA EG, PEREZ JF, MARTIN M, KAMEL C, BAUCELLS F AND GASA J. 2004. Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. J Anim Sci 82(11): 3210-3218. ) and mixture of fumaric and formic acids (Gomes et al. 2007GOMES FE, FONTES DO, SALIBA EOS, FERREIRA WM, FIALHO ET, SILVA FCO, SILVA MA, CORRÊA GSS AND SALUM GM. 2007. Ácido fumárico e sua combinação com os ácidos butírico ou fórmico em dietas de leitões recém-desmamados. Arq Bras Med Vet Zootec 59(5): 1270-1277. ).
The divergence in results due to usage of acidifiers in weaned piglets diets might be related to differences on raw material composition of experimental diets. Throughout this experiment, the diets had high inclusion of animal protein and lactose, which might be a substrate for microrganisms present in digestive tract, such as Lactobacillus spp., which produces lactic acid and decreases the pH. Owsley et al. (1988OWSLEY WF, HAYDON KD AND JONES RD. 1988. Effects of organic acid addition and diet complexity on performance of pigs weaned at 28 days. J Anim Sci 66(2): 1-41. ) observed that the use of simpler diets based on corn and soybean meal and low inclusion of dairy products have resulted on positive effects after acidifier inclusion. Other reasons for the divergence of results observed in researches performed with acidifiers are the palatability of diets that can inhibit or stimulate consumption, the buffering capacity of diets, the level of acidifier inclusion in diets and the animal’s age (Ravindran and Kornegay 1993RAVINDRAN V AND KORNEGAY ET. 1993. Acidification of weaner pig diets: a review. J Sci Food Agr 62(4): 313-322. ). In this study, the addition of organic acids in diets had no effect on palatability, since the feed intake was not different among treatments.
The addition of antibiotics and antimicrobial agents as copper sulfate and zinc oxide has also influenced the absence of responses from acidifiers (they might have masked the positive effects of organic acids), since they act similarly to those products (Katouli et al. 1999KATOULI M, MELIN L, JENSEN-WAERN M, WALLGREN P AND MÖLLBY R. 1999. Effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs. J Appl Microbiol 87(4): 564-573. ). Another important factor was high weight at weaning that might have contributed to reduce the organic acids responses, because piglets were healthier and mature (Radecki et al. 1988RADECKI SV, JUHL MR AND MILLER ER. 1988. Fumaric and citric acids feed additives in starter pig diets: effect on performance and nutrient balance. J Anim Sci 66(10): 2598-2605.).
The feed provided to piglets in farrowing stall benefits the development of digestive enzymes activity, improving diet utilization by weaned piglets (Lindemann et al. 1986LINDEMANN MD, CORNELIUS SG, EL KANDELGY SM, MOSER RL AND PETTIGREW JE. 1986. Effect of age, weaning and diet on digestive enzyme levels in the piglet. J Anim Sci 62(5): 1298-1307. ). The animals of this experiment received pre-starter diets in farrowing rooms, which might have contributed to the absence of acidifiers’ responses due to, according to Radecki et al. (1988RADECKI SV, JUHL MR AND MILLER ER. 1988. Fumaric and citric acids feed additives in starter pig diets: effect on performance and nutrient balance. J Anim Sci 66(10): 2598-2605.), this management practice can affect the efficiency of organic acids, once feed consumption in the farrowing house accelerates the maturation of piglets’ digestive system at weaning, promoting their greater adaptation.
The diarrhea, the most serious problems of weaned piglets, is characterized by loss of solutes and water, leading to depletion of electrolytes, acid-base imbalance and dehydration, which can be fatal if not treated properly (Zlotowski et al. 2008ZLOTOWSKI P, DRIEMEIER D AND BARCELLOS DESN. 2008. Patogenia das diarréias dos suínos: modelos e exemplos. Acta Sci Vet 36(1): S81-S86.). The use of additives, such as organic acids and their salts reduces or even eliminates the post-weaning diarrhea problems, acting in pathogenic bacteria control (Tsiloyiannis et al. 2001TSILOYIANNIS VK, KYRIAKIS SC, VLEMMAS J AND SARRIS K. 2001. The effect of organic acids on the control of porcine post-weaning diarrhoea. Res Vet Sci 70(3): 287-293. , Mazzoni et al. 2004MAZZONI M, LALATTA COSTERBOSA G, CASINI L, PETROSINO G, TREVISI P, DE FILIPPI S AND BOSI P. 2004. La morfologia dello stomaco come parametro di valutazione dell’impiego di acidificante nella dieta del suino in svezzamento. Società Italiana dele Scienze Veterinarie 56: 240. http://www.ismea.it/flex/AppData/Redational/Normative/D.1e1aaa02ed5d4f958508/200760651.pdf.
http://www.ismea.it/flex/AppData/Redatio...
, Bosi et al. 2005BOSI P, SARLI G, CASINI L, DE FILIPPI S, TREVISI P, MAZZONI M AND MERIALDI G. 2005. Effect of dietary addition of free or fat-protected calcium formate on growth, intestinal morphology and health of Escherichia coli K88 challenged weaning pigs. Ital J Anim Sci 4(2): 452-454. , Roselli et al. 2005ROSELLI M, FINAMORE A, BRITTI MS, BOSI P, OSWALD I AND MENGHERI E. 2005. Alternatives to in-feed antibiotics in pigs: Evaluation of probiotics, zinc or organic acids as protective agents for the intestinal mucosa. A comparison of in vitro and in vivo results. Anim Res 54: 203-218. , Freitas et al. 2006FREITAS LS, LOPES DC, FREITAS AF, CARNEIRO JC, CORASSA A, PENA SM AND COSTA LF. 2006. Avaliação de ácidos orgânicos em dietas para leitões de 21 a 49 dias de idade. Rev Bras Zootec 35(4): 1711-1719. ). No differences (P>0.05) were observed on diarrhea incidence among animals (Table III). Similar results reported by Risley et al. (1993RISLEY CR, KORNEGAY ET, LINDEMANN MD, WOOD CM AND EIGEL WN. 1993. Effect of feeding organic acids on gastrointestinal digesta measurements at various times postweaning in pigs challenged with enterotoxigenic Escherichia coli. Can J Anim Sci 73(4): 931-940. ) and Braz et al. (2011BRAZ DB, COSTA LB, BERENCHTEIN B, TSE MLP, ALMEIDA VV AND MIYADA VS. 2011. Acidificantes como alternativa aos antimicrobianos promotores do crescimento de leitões. Arch Zootec 60(231): 745-756. ) found no differences on diarrhea occurrence when diets were evaluated with combinations of organic acids for piglets. The fact that animals had been housed in pens subjected to a long empty period before the beginning of the experiment, although not desinfected, have contributed to a low health challenge what might explain the ineffectiveness of acidifier blend or fumaric acid on diarrhea reduction in piglets kept on that health challenge status.
EXPERIMENT 2
No FA x AB interaction effects neither differences on pH values of stomach, jejunum, ileum, colon and rectum (P>0.05) were registered (Table II). The stomach pH ranged from 3.10 to 3.68, which are within the range of pepsin action, that is 1 to 4 (Yamamoto 1975YAMAMOTO A. 1975. Proteolytic enzymes. In: Reed G (Eds), Enzymes in food processing, Holland: Elsevier, 2nd ed., p. 123-179. ). Gomes et al. (2011GOMES FE, FONTES DO, VASCONCELLOS CHF AND SILVA FCO. 2011. Ácido fumárico e sua combinação com ácido lático ou propionato de cálcio em dietas de leitões recém-desmamados. Arq Bras Med Vet Zootec 63(3): 678-686. ) found no statistical differences when added 0.5% fumaric acid and 0.5% fumaric acid plus 1% lactic acid in piglet diets and, reported gastric pH values of 4.13 and 4.23, respectively, for piglets at 38-day-old.
The absence of effects of AB on pH of stomach digesta (Risley et al. 1992RISLEY CR, KORNEGAY ET, LINDEMANN MD, WOOD CM AND EIGEL WN. 1992. Effects of feeding organic acids on selected intestinal content measurements at varying times postweaning in pigs. J Anim Sci 70(1): 196-206. , Freitas et al. 2006FREITAS LS, LOPES DC, FREITAS AF, CARNEIRO JC, CORASSA A, PENA SM AND COSTA LF. 2006. Avaliação de ácidos orgânicos em dietas para leitões de 21 a 49 dias de idade. Rev Bras Zootec 35(4): 1711-1719. , Bosi et al. 2007BOSI P, SARLI G, CASINI L, DE FILIPPI S, TREVISI P, MAZZONI M AND MERIALDI G. 2007. The influence of fat protection of calcium formate on growth and intestinal defence in Escherichia coli K88-challenged weanling pigs. Anim Feed Sci Technol 139: 170-185. , Braz et al. 2011BRAZ DB, COSTA LB, BERENCHTEIN B, TSE MLP, ALMEIDA VV AND MIYADA VS. 2011. Acidificantes como alternativa aos antimicrobianos promotores do crescimento de leitões. Arch Zootec 60(231): 745-756. ), duodenum pH (Bosi et al. 2007), jejunum pH (Risley et al. 1992) and ileum pH (Gabert and Sauer 1995GABERT VM AND SAUER WC. 1995. The effect of fumaric acid and sodium fumarate supplementation to diets for weanling pigs on amino acid digestibility and volatile fatty acid concentration in ileal digesta. Anim Feed Sci Technol 53: 243-254. , Bosi et al. 2007) was similar to the observed in this experiment, suggesting that this is not the most efficient mode of action for acidifiers’ usage. The differences on pH values found in segments of digestive tract might occur depending on the location where measurements were taken, the methodology used and the time after eating (Ferreira 1986FERREIRA AS. 1986. Estimativa de Produção e Composição de Leite de Porca e Aleitamento Artificial de Leitões. Tese de Doutorado. Viçosa: Universidade Federal de Viçosa.). In the present study, sampling was performed after the slaughter of animals, which did not undergo fasting. Another factor that might have influenced the response of acidifier was the lactose inclusion in diets, which is used as substrate for Lactobacillus spp. to produce lactic acid, which in turn reduces the pH.
However, some researches have shown acidifiers efficiency in reducing pH of stomach digesta (Thomlinson and Lawrence 1981THOMLINSON JR AND LAWRENCE TL. 1981. Dietary manipulation of gastric pH in the prophylaxis of enteric disease in weaned pigs: some field observations. Vet Rec 109(6): 120-122. , Bolduan et al. 1988BOLDUAN VG, JUNG H, SCHNEIDER R, BLOCK J AND KLENKE B. 1988. Influence of propionic- and formic acid on piglets. J Anim Physiol Anim Nutr 59: 72-78., Maribo et al. 2000MARIBO H, JENSEN BB AND HEDEMANN MS. 2000. Different doses of organic acids for weaned piglets. In: The National Committee for Pig Production, Denmark: Danish Bacon and Meat Council, report 469.), cecum pH (Maribo et al. 2000) and colon pH (Maribo et al. 2000, Namkung et al. 2004NAMKUNG H, LI M, GONG J, YU H, COTTRILL M AND LANGE CFM. 2004. Impact of feeding blends of organic acids and herbal extracts on growth performance, gut microbiota and digestive function in newly weaned pigs. Can J Anim Sci 84(4): 697-704. ). The reduction on digesta pH limits the development of pathogenic microrganisms, such as Escherichia coli, which does not survive in acidic conditions (Barcellos et al. 2007BARCELLOS DESN, SOBESTIANSKY J AND SOBESTIANSKY T. 2007. Uso de antimicrobianos. In: Sobestiansky J and Barcellos DESN (Eds), Doenças dos Suínos. Goiânia: Cânone Editora, p. 685-717.), but usually grows in a pH range from 5 up to 9 (Zilberstein et al. 1984ZILBERSTEIN D, AGMON V, SCHULDINER S AND PADAN E. 1984. Escherichia coli intracellular pH, membrane potential, and cell growth. J Bacteriol 158(1): 246-252.).
No FA x AB interaction or main factors effects were observed (P>0.05) on relative weights of stomach, small intestine, cecum, liver, spleen and kidneys. However, the addition of FA in diet determined lower relative weight of pancreas (P = 0.009) and, the addition of AB resulted on higher relative weight of large intestine (P = 0.009) (Table IV). Similar results were reported by Bhandari et al. (2008BHANDARI SK, XU B, NYACHOTI CM, GIESTING DW AND KRAUSE DO. 2008. Evaluation of alternatives to antibiotics using an Escherichia coli K88+ model of piglet diarrhea: effects on gut microbial ecology. J Anim Sci 86(4): 836-847. ), which added the mixture of organic acids in piglet diets and did not observe effect on spleen relative weight, because this organ weight varies depending on the energy and/or protein intake (Rao and McCracken 1992RAO DS AND MCCRACKEN KJ. 1992. Energy: protein interactions in growing boars on high genetic potential for lean growth. 1. Effects on growth, carcass characteristics and organ weights. Anim Prod 54(1): 75-82. ). The diets used in this experiment were isoproteic and had similar levels of metabolizable energy, which could justify the fact that no differences were found on relative weights of stomach, small intestine, cecum, liver, spleen and kidneys.
No FA x AB interaction effects and no effects of main factors (P>0.05) were observed on villi height (VH) of duodenum, villi width (VW), crypt depth (CD), villi height:crypt depth ratio (VH:CD), and absorption areas of duodenum and jejunum (Table V). The jejunum villi height of piglet fed diet containing fumaric acid was lower (P = 0.009) compared to animals fed diet without FA; however, the jejunal absorption area (Table V) was similar, indicating that presence of FA in diet resulted in an increase on villi density.
After evaluate FA inclusion of 0.5 to 1% in diets, Gomes et al. (2007GOMES FE, FONTES DO, SALIBA EOS, FERREIRA WM, FIALHO ET, SILVA FCO, SILVA MA, CORRÊA GSS AND SALUM GM. 2007. Ácido fumárico e sua combinação com os ácidos butírico ou fórmico em dietas de leitões recém-desmamados. Arq Bras Med Vet Zootec 59(5): 1270-1277. ) found no effects on small intestine morphometric characteristics. In contrast, Bosi et al. (2007BOSI P, SARLI G, CASINI L, DE FILIPPI S, TREVISI P, MAZZONI M AND MERIALDI G. 2007. The influence of fat protection of calcium formate on growth and intestinal defence in Escherichia coli K88-challenged weanling pigs. Anim Feed Sci Technol 139: 170-185. ) reported greater villi height values in small intestine of piglets weaned at 21-day-old when calcium formate were provided in diets.
The reduction on intestinal villi height of weaned piglets occur at 3rd to 7th days after weaning (Cera et al. 1988CERA KR, MAHAN DC, CROSS RF, REINHART GA AND WHITMOYER RE. 1988. Effect of age, weaning and post-weaning diet on small intestinal growth and jejunal morphology in young swine. J Anim Sci 66(2): 574-584. ) and, the intensity of intestinal disorders are more associated with ingredients used in diet formulation (Li et al. 1990LI DF, NELSSEN JL, REDDY PG, BLECHA F, HANCOCK JD, ALLEE GL, GOODBAND RD AND KLEMM RD. 1990. Transient hypersensivity to soybean meal in the early-weaned pig. J Anim Sci 68(6): 1790-1799.) and the quantity of ingested feed (Van Beers-Schreurs et al. 1998). The decrease on villi height are mainly due to the increase in cell desquamation rate (Thomaz et al. 2002THOMAZ MC, TUCCI FM AND KRONKA RN. 2002. Efeitos da adição de glutamina, ácidos graxos poliinsaturados ou parede celular de levedura na dieta de leitões desmamados sobre a estrutura do intestino delgado. In: Reunião Anual da Sociedade Brasileira de Zootecnia, Recife. Proceedings.... Campinas: ABZ. ) which predisposes animals to nutrient malabsorption, diarrhea and dehydration (Scandolera et al. 2005SCANDOLERA AJ, THOMAZ MC, KRONKA RN, FRAGA AL, BUDIÑO FEL, ROBLES-HUAYNATE RA, RUIZ US AND CRISTANI J. 2005. Efeitos de fontes protéicas na dieta sobre morfologia intestinal e desenvolvimento pancreático de leitões recém-desmamados. Rev Bras Zootec 34(6): 2355-2368. ). Higher VH:CD ratios indicate the presence of increased amount of mature and functional enterocytes, which results in better digestion and absorption of nutrients and also, minor injuries in intestinal mucosa (Tucci et al. 2011TUCCI FM, THOMAZ MC, NAKAGHI LSO, HANNAS MI, SCANDOLERA AJ AND BUDIÑO FEL. 2011. Efeito da adição de agentes tróficos na dieta de leitões desmamados sobre a estrutura e ultraestrutura do intestino delgado e sobre o desempenho. Arq Bras Med Vet Zootec 63(4): 931-940. ).
The animals were kept during the experimental period in a controlled environment, under good conditions of temperature and hygiene, as well as receiving pre-starter diets with high inclusion of lactose, which help to reduce the stress of weaning, contributing to minor changes of intestinal mucosa (Figueiredo et al. 2003FIGUEIREDO AN, MIYADA VS, UTIYAMA CE AND LONGO FA. 2003. Ovo em pó na alimentação de leitões recém-desmamados. Rev Bras Zootec 32(6): 1901-1911., Thomaz et al. 2011THOMAZ MC, HANNAS MI, KRONKA RN, TUCCI FM, SCANDOLERA AJ AND BUDIÑO FEL. 2011. Plasma suíno e ovo inteiro em rações de leitões desmamados sobre a estrutura e ultra-estrutura da muscosa intestinal. Ars Vet 27(4): 246-254. ).
The nutrient absorption capacity is proportional to density and size of the villi (Macari 1995MACARI M. 1995. Mecanismos de proliferação e reparação da mucosa gastrintestinal em aves. In: Simpósio de Coccidiose e Enterite, 1. Proceedings … Campinas: [s.n.]. ). Skrzypek et al. (2010SKRZYPEK T, VALVERDE PIEDRA JL, SKRZYPEK H, KAZIMIERCZAK W, SZYMANCZYK SE AND ZABIELSKI R. 2010. Changes in pig small intestinal absorptive area during the first 14 days of life. Livest Sci 133: 53-56. ) used piglets at 14-day-old and the mean values obtained from duodenum and jejunum absorption area of 9.3 mm²/mm2 and 14.4 mm²/mm², respectively were higher than those found in this study for piglets of 35-day-old; although they reported that gut absorption area increased rapidly until 7th day of age, and reduced on 14th day of piglets’ age.
The use of ingredients with anti-nutritional factors and/or antigenic protein in diet causes changes on intestinal mucosa, including damage to villi and impair absorption of nutrients (Chamone et al. 2010CHAMONE JMA, MELO MTP, AROUCA CLC, BARBOSA MM, SOUZA FA AND SANTOS D. 2010. Fisiologia digestiva de leitões. Nutritime 7(5): 1353-1363.). The diets used in this experiment were formulated, mostly using highly digestible ingredients, which may have contributed to absence of acidifier effects for most of the variables.
No FA x AB interaction effects were observed on total coliform counts (TC) and Lactobacillus counts (LC) (Table VI). However, animals fed diet containing FA showed lower (P<0.05) total coliform and Escherichia coli (EC) counts than piglets fed diet without FA, proving the antimicrobial capacity of the acid. The basic principle of FA action is related to its undissociated forms (non-ionized, lipophilic form) that diffuse across cell membranes of bacteria, inhibiting their growth through inactivation of bacterial decarboxylases and catalases or even though, over long periods of exposure to FA, desrupting their cell wall (due to an excess of intracellular H+ ions) and/or also to the intestinal lumen dissociation that liberates H+ ions serving as a pH barrier against bacteria colonization on brush border (Suiryanrayna and Ramana 2015SUIRYANRAYNA MVAN AND RAMANA JV. 2015. A review of the effects of dietary organic acids fed to swine. J Anim Sci Biotechnol 6(45): 1-11.). Although, after a reduction on total coliform counts, we also expected a reduction on Escherichia coli counts, once these microrganisms constitute the major part of enterobacteria population, the results obtained in this study were contrary to those reported by Risley et al. (1992RISLEY CR, KORNEGAY ET, LINDEMANN MD, WOOD CM AND EIGEL WN. 1992. Effects of feeding organic acids on selected intestinal content measurements at varying times postweaning in pigs. J Anim Sci 70(1): 196-206. ), which found no reduction on Escherichia coli counts in cecum of weaned piglets fed diet containing 1.5% FA. These results agreed with results of studies in which the presence of acidifier has determined a reduction on Escherichia coli counts in faeces (Tsiloyiannis et al. 2001TSILOYIANNIS VK, KYRIAKIS SC, VLEMMAS J AND SARRIS K. 2001. The effect of organic acids on the control of porcine post-weaning diarrhoea. Res Vet Sci 70(3): 287-293. , Knarreborg et al. 2002KNARREBORG A, MIQUEL N, GRANLI T AND JENSEN BB. 2002. Establishment and application of an in vitro methodology to study the effects of organic acids on coliform and lactic acid bacteria in the proximal part of the gastrointestinal tract of piglets. Anim Feed Sci Technol 99: 131-140.).
The lack of responses regarding the acidifier blend usage confirms the results reported by Walsh et al. (2007WALSH MC, SHOLLY DM, HINSON RB, TRAPP SA, SUTTON AL, RADCLIFFE JS, SMITH JW AND RICHERT BT. 2007. Effects of acid LAC and Kem-Gest acid blends on growth performance and microbial shedding in weanling piglets. J Anim Sci 85(2): 459-467. ), that evaluated a combination of organic acids and did not observe reduction on Escherichia coli counts in weaned piglets fecal content, as well as those described by Torrallardona et al. (2007TORRALLARDONA D, CONDE R, BADIOLA I AND POLO J. 2007. Evaluation of spray dried animal plasma and calcium formate as alternatives to colistin in piglets experimentally infected with Escherichia coli K99. Livest Sci 108: 303-306. ), that analyzed inclusion of 1.8% of calcium formate in diets and found no difference on Escherichia coli counts in cecum and ileum of weaned piglets compared to other treatments. Bhandari et al. (2008BHANDARI SK, XU B, NYACHOTI CM, GIESTING DW AND KRAUSE DO. 2008. Evaluation of alternatives to antibiotics using an Escherichia coli K88+ model of piglet diarrhea: effects on gut microbial ecology. J Anim Sci 86(4): 836-847. ), working with piglets weaned at 17-day-old and evaluating combinations of organic acids in diets, found no difference on Escherichia coli counts in ileum and cecum among treatments, highlighting that low number of replicates and inherent variability at post-weaning might have caused the lack of answers. Moreover, lack of responses on the reduction of total coliform and Escherichia coli counts might also be explained by the low AB inclusion (0.3%) in experimental diets.
CONCLUSIONS
The inclusion of fumaric acid and the acidifier blend in diets containing colistin or halquinol is not indicated for weaned piglets from 21- to 62-day-old, regarding the growth performance results, pH values of digesta, relative weight of organs and intestinal morphometry. Also, the fumaric acid exerted inhibitory action on growth of total coliform and Escherichia coli population in piglets’ cecum.
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Publication Dates
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Publication in this collection
Jan-Mar 2018
History
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Received
01 Feb 2016 -
Accepted
13 July 2016