research article effects of dietary supplementation of oregano...

Research ArticleEffects of Dietary Supplementation of Oregano Essential Oil toSows on Oxidative Stress Status Lactation Feed Intake of Sowsand Piglet Performance

Chengquan Tan1 Hongkui Wei1 Haiqing Sun2 Jiangtao Ao1 Guang Long1

Siwen Jiang3 and Jian Peng1

1Department of Animal Nutrition and Feed Science College of Animal Science and Technology Huazhong Agricultural UniversityWuhan 430070 China2YangXiang Joint Stock Company Guigang 53700 China3Key Laboratory of Swine Breeding and Genetics of the Agricultural Ministry College of Animal Science and TechnologyHuazhong Agricultural University Wuhan 430070 China

Correspondence should be addressed to Jian Peng pengjianmailhzaueducn

Received 9 June 2015 Revised 31 August 2015 Accepted 7 September 2015

Academic Editor M Hasan Mohajeri

Copyright copy 2015 Chengquan Tan et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fifty-four multiparous large white sows were used to determine the effects of supplementing oregano essential oil (OEO) to thegestation and lactation diets on oxidative stress status lactation feed intake and their piglet performance Two groups were feddiets with (OEO 119899 = 28) or without (Control 119899 = 26) supplemental 15mgkg OEO during gestation and lactation The serumlevels of reactive oxygen species (ROS) (119875 lt 005) 8-hydroxy-deoxyguanosine (8-OHdG) (119875 lt 005) and thiobarbituric acidreactive substances (TBARS) (119875 lt 005) were higher during gestation (days 90 and 109) and lactation (days 1 and 3) than in earlygestation (day 10) Compared with the control group the OEO diet significantly reduced sowsrsquo serum concentrations of 8-OHdG(119875 lt 005) and TBARS (119875 lt 001) on day 1 of lactation The OEO diet increased the sowsrsquo counts of faecal lactobacillus (119875 lt 0001)while reducing Escherichia coli (119875 lt 0001) and Enterococcus (119875 lt 0001) In the third week of lactation the treatment tended toincrease sowrsquos feed intake (119875 = 007) which resulted in higher average daily gain (119875 lt 001) of piglets Our results demonstratedthat there is an increased systemic oxidative stress during late gestation and early lactation of sows The OEO supplementation tosowsrsquo diet improved performance of their piglets which may be attributed to the reduced oxidative stress

1 Introduction

Oxidative stress results from increased production of reactiveoxygen species (ROS) or a decrease in antioxidant defenseOxidative damage is a strong indicator of health status andwellbeing of animals [1] A recent study showed that pregnantsows had elevated oxidative stress during late gestationand lactation [2] which was responsible for impaired milkproduction reproductive performance and finally longevityof sows [3ndash5] Accumulated evidence suggests that excessiveROS affect the insulin signaling cascade which leads toinsulin resistance [6 7] Insulin resistance during peripartalperiod was shown to have a negative effect on lactation feedintake of sows [8 9]Thus dietary antioxidant concentrations

need to be reevaluated for their sufficiency in sow diets espe-cially to prevent excessive oxidative stress during gestationand lactation

Oregano essential oil (OEO) is isolated from plants (Ori-ganum vulgare L) by steam distillation Chemical analyses ofthese oils have shown the principal nutraceutical constituentsto be carvacrol and thymol [10] In vitro OEO has beenreported to possess antimicrobial [11 12] and antioxidantactivities [13 14] Although previous studies have reportedthat dietary supplementation of OEO to sows reduced thefat percentage in milk did not affect growth pattern ofsuckling pigs and increased reproductive performance ofsows [15ndash17] its effect on sowsrsquo oxidative stress status duringgestation and lactation remains unknown Therefore the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 525218 9 pageshttpdxdoiorg1011552015525218

2 BioMed Research International

objective of this study was to examine the effects of dietarysupplementation of sow diets with OEO during gestationand lactation on oxidative stress status colostrum and milkcomposition lactation feed intake and piglet performance

2 Materials and Methods

All experimental procedures were approved by the AnimalCare and Use Committee of Huazhong Agricultural Univer-sity

21 Animals Diet Treatments and Management Sixty largewhite sowswere originally allotted to the study the sowsweredivided into two groups (control group andOEOgroup) of 30animals After breeding six sows (4 in control group and 2 inOEO group) were returned to estrus within the estrus cycleFifty-four large white sows and parities of 495 plusmn 112 wereused in this trial After breeding the sows were randomlyallotted to 1 of 2 experimental dietary treatments based onparity and body weight (BW) control sows were fed a basaldiet (Table 1) with no supplementation (C 119899 = 26) and thetreatment sows were fed a basal diet added with 15mgkgOEO through gestation and lactation (OEO 119899 = 28)The commercially available OEO is a mixture powder thatcontains 5 OEO of Origanum vulgare subsp hirtum plantsand 95 natural feed grade inert carrier For each kilogramof sow feed we supplemented 300mg Orego-Stim (MeridenAnimalHealth Ltd UK) that is 15mgOEOThe componentsof OEO were shown in Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552015525218 TheOEO supplement contained carvacrol (8192) and thy-mol (350) Sows from the two groups were restrict-fedwith their respective diets during gestation Sows were fed20 kgday from days 1 to 30 of gestation 25 kgday from days31 to 90 of gestation and 30 kgday from day 91 of gestationto farrowing The diets were supplied twice a day (0700 and1430) During lactation the diet was supplied three times aday (0700 1100 and 1730) to ensure sows ad libitum accessto feed Pregnant sows were housed individually in gestationstalls (22m times 07m times 11m) Sows were moved from thegestation stalls to the farrowing rooms on day 107 plusmn 2 ofgestation and then kept in individual farrowing crates withstalls (22m times 07m) in pens that provided space on bothsides of the stall (22m times 05m) for the pigs after birthBoth sows and piglets had free access to water Piglets werenot offered creep feed Sow milk was the only feed availableto the piglets during lactation During the experimentalperiod data from sows with illness serious lameness deathand reproductive failure were not included in the analyses(Table 2)

22 Performance Measurement BW and backfat thicknessof sows were measured on days 0 and 107 of pregnancywithin 24 h of farrowing and at weaning Backfat thicknessat 65mm on each side of the dorsal midline at the last rib(1198752) was measured using ultrasound (PIGLOG105 SFAK-

Technology) At farrowing the numbers of total pigletsborn and piglets born alive were recorded The piglets werecross-fostered within dietary treatment groups by 48 h after

Table 1 Composition of the gestation and lactation diets (as-fedbasis)

Item Gestation LactationIngredient Corn 5630 5440Soybean meal 43 CP 1000 2600Wheat bran 3040 1100Calcium carbonate 120 151Dicalcium phosphate 104 123Salt 040 026Mildewcide1 012 010Choline chloride 014 100Premix2 040 150

Nutrient compositionNet energy MJkg3 932 1036Crude protein 1406 1892Lysine 3 061 103Calcium 3 077 106Available phosphorus 3 033 045

1Mildewcide ammonium propionate2Provided per kg of diet Cu 30mg Fe 160mg Zn 160mg Mn 55mg I05mg Se 05 Co 08mg Cr 02mg Vitamin A 14000 IU Vitamin D32900 IU Vitamin E 120mg Vitamin K3 6mg Vitamin B1 24mg VitaminB2 85mg Vitamin B6 45mg Vitamin B12 003mg Vitamin H 055mgPantothenic acid 30mg Folic acid 5mg Nicotinamide 50mg3Calculated chemical concentrations using values for feed ingredients fromthe National Research Council (1998)

Table 2 The number of sows during the experimental periods

Item C1 OEO1

Breeding 26 28Culled during gestation2 3 1Parturition 23 27Culled during lactation2 1 2Weaning 22 251Dietary treatments C = control diet OEO = 15mgkg oregano essential oilsdiet2Data of sows that were ill seriously lame died during the study and hadreproductive failure were not included

farrowing to adjust the litter size The number of piglets persow ranged from 9 to 12 piglets At weaning the numbers ofweaned piglets were recorded Piglets were weighed within24 h of birth (day 1) and on days 7 14 and 21 The daily feedintake of sows during lactation was recorded each morningby weighing daily feed refusals

23 Samples Collection At 2 h after feeding on days 10 6090 and 109 of gestation and on days 1 3 7 and 21 of lactationbefore feeding on days 10 and 109 of gestation and on days 3and 7 of lactation blood samples were collected from sows (5sows per diet group with the similar parity) by ear vein witha minimum amount of stress into heparinized tubes (5mL)or in tubes containing no anticoagulant (5mL) Fasting sowswere selected for blood sampling after an overnight fastingperiod of 16 h during gestation and 12 h during lactation

BioMed Research International 3

Table 3 Species and genus specific primers used for real time PCR to profile selected bacteria

Target group Sequence of primers (51015840-31015840) Product size (bp) Annealing temperature (∘C)

Escherichia coli CATGCCGCGTGTATGAAGAA 96 60CGGGTAACGTCAATGAGCAAA

Enterococcus CCCTTATTGTTAGTTGCCATCATT 144 61ACTCGTTGTACTTCCCATTGT

Lactobacillus AGCAGTAGGGAATCTTCCA 341 58CACCGCTACACATGGAG

Samples collected for plasma assays (heparinized tubes) werekept on ice and centrifuged for 5min at 8500timesg at 4∘CSamples for serum assays (tubes containing no anticoagulant)were left at room temperature for 4 h and then centrifugedfor 5min at 5000timesg at 4∘C Serum and plasma samples werestored at minus80∘C until they were assayed Colostrum samples(30mL) were collected from the third fourth and fifth pairsof mammary glands of sows (5 sows per diet group with thesimilar parity) within 4 h after the initiation of farrowingMilk samples (30mL) were also collected from the thirdfourth and fifth pairs of mammary glands of sows (5 sowsper diet group with the similar parity) on day 18 after anintramuscular injection of 10 IU oxytocin behind an ear Thecolostrum and milk samples were immediately frozen atminus20∘Cuntil analysis Fresh faecal sampleswere collected fromthe sows (5 sows per diet groupwith the similar parity) on day109 of gestation into individual plastic containers and keptfrozen at minus20∘C

24 Quantification of Faecal Bacteria Bacterial DNA wasextracted and purified from faeces samples using a QIAampDNA stool kit (Qiagen Germany) in accordance with themanufacturerrsquos instructions Genomic DNA from faeces waspooled and amplified through routine PCR using speciesand genus specific primers (Table 4) After PCR amplificationwith a Taq DNA polymerase kit (Promega USA) and elec-trophoresis on a 15 agarose gel PCR products were purifiedaccording to the manufacturerrsquos protocol (Omega USA)Thepurified PCR products were linked to the pMD18-Tvectorsystem (Takara Bio Inc) and then transferred to Escherichiacoli DH5120572 (Qiagen Germany) to clone After checkingthe size of the cloned inserts with PCR amplification theextracted plasmids of the positive clones were sequencedcommercially obtaining the positive plasmids

Serial dilutions of these positive plasmids served togenerate standard curves using quantitative real time PCR(BIO-RAD System USA) permitting estimations of absolutequantification based on respective gene copies After 10-folddilution microbial genomic DNAwas performed to estimateabsolute quantificationThe reactionwas performed in a totalvolume of 20 120583L containing 4 120583L template DNA 1 120583L forwardand reverse primers 10 120583L iTaq SYBR Green PCR MasterMix (BIO-RAD USA) and 5 120583L nuclease-free water Thethermal cycling conditions involved an initial denaturationstep at 95∘C for 4min followed by forty cycles of 95∘C for 10 sannealing temperature (Table 3) for 10 s and 72∘C for 30 sfollowed by a productmelting curve to confirm the specificityof amplification The mean threshold cycle values from the

Table 4 Effects of dietary supplementation of oregano essentialoils to sows in gestation and lactation on the colostrum and milkcomposition and IgG and IgM concentrations in colostrum andserum of sows

Item C1 OEO1 SEM 119875 valueNumber of sows 5 5Colostrum2

Fat () 578 460 036 014Lactose () 198 201 007 084Protein () 1502 1588 060 051Solid not fat () 2050 2134 053 047Total solids () 2710 2658 073 075IgG (mgmL) 1580 1579 046 091IgM (mgmL) 286 285 014 097

Milk ()3 d 18 of lactationFat 871 810 056 061Lactose 484 479 012 085Protein 456 460 006 077Solid not fat 1360 1359 011 097Total solids 2218 2164 040 053

Serum d 109 of gestationIgG (mgmL) 890 782 046 027IgM (mgmL) 269 282 015 069

SEM standard error of means1Dietary treatments C = control diet OEO = 15mgkg oregano essential oilsdiet2Colostrum was collected within 4 h after the initiation of farrowing3Sows were injected with 10 IU of oxytocin intramuscularly behind the earto induce milk ejection

triplicate of each sample were used for calculations The datawas calculated as gene copy numbers per gram of wet faecesand presented as Log

10CFUg faeces for the convenience of

data analysis

25 Analysis of Oxidative Stress Parameters Serum sam-ples were used to measure levels of thiobarbituric acidreactive substances (TBARS) 8-hydroxy-deoxyguanosine (8-OHdG) glutathione peroxidase (GSH-Px) and reactiveoxygen species (ROS) An uncontrolled increase in ROSproduction leads to peroxidative damage of macromoleculeswhich in turn may cause disturbances in the metabolismand physiology [18] TBARS is one of the most frequentlyused indicators of lipid peroxidation and was determined inthe current study The major marker for oxidative damage tonucleic acids 8-OHdG was chosen to determine the DNA


damage in the current study [19] Serum samples were ana-lyzed for activities of antioxidant enzymes including GSH-Px and for TBARS using the commercial kits provided byNanjing Jiancheng Bioengineering Institute (Nanjing China)[20] GSH-Px activity was determined based on quantifyingthe rate of oxidation of GSH to GSSG by H

2O2catalyzed by

GSH-Px GSH reacts with 551015840-dithiobis-p-nitrobenzoic acid(DTNB) to produce yellow colored 5-thio-2-nitrobenzoicacid (TNB) that can be quantified spectrophotometrically at412 nm TBARS was analyzed based on the reaction with 2-thiobarbituric acidThe resulting pink product wasmeasuredspectrophotometrically at 535 nm An ELISA kit (DobioBiotech Co LTD Shanghai China) that utilizes an anti-8-OHdGmonoclonal antibody to recognize 8-OHdG was usedto determine the concentration of 8-OHdG in the serumsample according to the method described by Pialoux etal [21] Levels of ROS were measured in serum by chemi-luminescence assay using luminol (5-amino-23-dihydro-14-phthalazinedione Sigma) as probe The measurementsaccording to procedure were described in detail by Du et al[22]

26 Laboratory Analyses Crude protein was determinedaccording to AOAC (1990) The milk composition wasdetermined with a near infrared reflectance spectroscopymethod by Milk-Scan 134AB Immunoglobulin concentra-tions were assessed in serum (IgG and IgM) and mammary(IgG and IgM) secretions by ELISA using pig polyclonalimmunoglobulin-specific kits (Bethyl Montgomery USA)Prior to analysis colostrum and milk were delipidated bycentrifugation at 3000timesg at 4∘C for 20min Plasma concen-trations of glucose and insulin were determined accordingto the glucose dehydrogenase activity colorimetric assay kit(BioVision Inc CA USA) and insulin ELISA kit (BiosourceInc Sunnyvale CA USA) according to the manufacturerrsquosinstructions respectively All samples were analyzed in dupli-cate The indirect methods were used to evaluate insulin sen-sitivity by homeostasis model assessment (HOMA) HOMA-IR (insulin resistance) = [(fasting insulin mIUL)] times (fastingglucose mmolL)]225 HOMA-IS (insulin sensitivity) =1[(fasting insulin mIUL)] times (fasting glucose mmolL)][23]

27 Statistical Analyses An individual sow was consideredthe experimental unit in all statistical analyses Results wereanalyzed by ANOVA using the general linear model proce-dure (SAS 80 Inst Inc Cary NC) For sows and litter per-formances the model included the effects of treatment andreplicate and their interaction The number of total pigletsborn was used as a covariate in the analysis of piglet birthweight and total litter weight at birth The piglet weight andlitter weight on day 21 of lactation were subjected to analysisof covariance with the piglet weight and litter weight aftercross-foster as the covariate Variations of oxidative stressparameters and HOMA values were analyzed by ANOVAusing the procedure for repeated measurements of SAS Themodel included the effects of treatment physiological stageand replicate When an interaction was significant this wasspecified in the text Data were given as means and SEM

Differences between treatment means were significant at 119875 lt005 and trends identified when 119875 gt 005 but lt010

3 Results

31 Oxidative Stress Parameters Faecal Microbial Counts andHOMA Values of Sows Serum levels of GSH-Px TBARS 8-OHdG and ROS on different days of gestation and lactationare shown in Figure 1 There was a treatment times sampling dayinteraction for serum TBARS concentrations (119875 lt 005) Theresults showed that in both groups serum levels of ROS andTBARS were higher (119875 lt 005) during late gestation (days 90and 109) and lactation (days 1 and 3) than in early gestation(day 10) Additionally in both groups serum concentrationsof 8-OHdGwere higher (119875 lt 005) during gestation (days 6090 and 109) and lactation (days 1 3 7 and 21) than in earlygestation (day 10) Compared with the C group sows underOEO treatment had significantly lower serum concentrationsof TBARS (119875 lt 001) and 8-OHdG (119875 lt 005) on day 1 oflactation They tended to have higher serum concentrationsof GSH-Px on day 60 of gestation (119875 = 008) and day 1 (119875 =007) of lactation lower serum concentrations of 8-OHdG onday 109 of gestation (119875 = 009) and day 3 of lactation (119875 =009) and also lower serum levels of ROS on day 1 (119875 = 009)and day 3 (119875 = 008) of lactation than sows fed C diet Inaddition the OEO diet significantly increased the counts offaecal Lactobacillus (119875 lt 0001) whereas it reduced the countsof Escherichia coli (119875 lt 0001) and Enterococcus (119875 lt 0001)on day 109 of gestation (Figure 2) It was also found that theOEO diet tended to reduce the value of HOMA-IR (119875 = 007)but increased the value of HOMA-IS (119875 = 006) of the sowson day 109 of gestation (Figure 3)

32 Colostrum and Milk Composition and IgG and IgMConcentrations in Colostrum and Serum of Sows Table 4showed that the dietary treatments had no effect on thecolostrum and milk composition as well as IgG and IgM inserum and colostrum

33 Sow Performance OEOdietary supplementation of sowsduring gestation and lactation did not affect the BW andbackfat gain during gestation lactation weight backfat lossor weaning-to-estrus of sows (Table 5) Sows in the OEOtreatment group tended to increase feed intake in the thirdweek of lactation in comparison to the C group (646 versus603 kgday 119875 = 007) (Table 5)

34 Pigletrsquos Performance The effect of the dietary treatmenton piglet performance is shown in Table 6 There were nodifferences in the numbers of total piglets born live-bornand weaned However sows fed the OEO diet significantlyincreased average piglet weights at birth (156 versus 144 kg119875 = 004) and on day 21 of lactation (694 versus 649 kg119875 = 001) Furthermore average daily gain (ADG) of pigletsduring the third week (30651 versus 27312 gd 119875 lt 001)and on days 1ndash21 of lactation (25236 versus 23361 gd 119875 lt001) were significantly increased for sows in the OEO dietgroup


AB A AB AB B AB AB B

Gestation Lactation

COEO

0

200

400

600G

SH-P

x (U

mL)

G 109G 90G 60 L 21L 1 G 10 L 3 L 7

(a)

COEO

lowast

C C B AB A B C C

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

1

2

3

4

TBA

RS (n

mol

mL)

(b)

COEO

C B AB AB AB A

lowast

AB AB

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

10

20

30

40

50

8-O

HdG

(ng

mL)

(c)

COEO

E E B B A C DE CD

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

2000

4000

6000

ROS

(RLU

)

(d)

Figure 1 Diet effects on serum levels of GSH-Px (a) TBARS (b) 8-OHdG (c) and ROS (d) of sows (means plusmn SEM 119899 = 5) AndashEEffect ofsampling day (119875 lt 005) Effect of dietary treatment (119875 lt 01) lowastEffect of dietary treatment (119875 lt 005)There was a treatment times sampling dayinteraction for serum TBARS concentrations (119875 lt 005) C = control diet OEO = 15mgkg oregano essential oils diet GSH-Px glutathioneperoxidase TBARS thiobarbituric acid reactive substances ROS reactive oxygen species 8-OHdG 8-hydroxy-deoxyguanosine RLU relativelight units

4 Discussion

The peripartal period particularly the delivery is a criticaltime for maintaining a balance between the production offree radicals and the incompletely developed antioxidativeprotection of the fetus and the newborn [24] Lipid perox-idation and antioxidant status are changed during deliveryand these changes affect the fetus by creating oxidative stress[25 26] Our study indicated that not only during delivery butalso during late gestation and early lactation the sows sufferfrom increased oxidative stress indicated by their elevatedROS 8-OHdG and TBARS levels These results were similarto a report from Berchieri-Ronchi et al [2] which showedthat there was an increased systemic oxidative stress during

gestation and lactation and that the sows were not fullyrecovered until weaning

In our study the OEO diet significantly reduced theconcentrations of both TBARS and 8-OHdG on day 1 oflactation One possible explanation is that sows suffer fromthe greatest oxidative stress then In the other two parameters(GSH-Px and ROS) positive effects of supplementing OEOwere also found This is in line with the previous study[27] reports in weaned pigs This positive effect of OEOcould probably be attributed to its composition whichmainlycontained carvacrol (8192) and thymol (350) (Table S1)because both carvacrol and thymol have been reported toscavenge superoxide radicals and hydrogen peroxide [28 29]


E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es

Figure 2 Diet effects on faecal bacterial counts (Log10cfug) on day

109 gestation of sows Values are means plusmn SEM (119899 = 5) lowastSignificantdifference between groups 119875 lt 0001 C = control diet OEO =15mgkg oregano essential oils diet E coli Escherichia coli EnteroEnterococcus Lacto Lactobacillus CFU colony forming unit

Table 5 Effects of dietary supplementation of oregano essential oilsto sows in gestation and lactation on sow performance

Item C1 OEO1 SEM 119875 valueNumber of sows 22 25Daily allowances during gestation kgd 242 242 001 099Sow BW kg

Breeding 2327 2340 401 059Gestation day 107 2673 2681 331 088Gain 346 341 208 033Parturition 2463 2473 330 098Weaning 2363 2370 340 075Loss 100 103 154 045

Sow backfat thickness mmBreeding 162 160 043 078Gestation day 107 170 175 047 088Gain 08 16 035 079Parturition 163 170 049 046Weaning 144 141 037 056Loss 19 29 034 046

Average daily feed intake kg1st week of lactation 421 417 015 0882nd week of lactation 593 590 015 0883rd week of lactation 603 646 017 007Mean of 1st week to 3rd week 539 551 013 055

WEI d 489 463 011 021SEM standard error of means BW body weight WEI weaning-to-estrusinterval1Dietary treatments C = control diet OEO = 15mgkg oregano essential oilsdiet

Table 6 Effects of dietary supplementation of oregano essential oilsto sows in gestation and lactation on piglet performance

Item C1 OEO1 SEM 119875 valueNumber of sows 22 25Litter size numberlitterTotal born 1159 1128 051 065Born alive 1141 1116 052 071After cross-foster 1000 976 017 050Pigs weaned 945 960 018 070

Litter weight kgAt birth 1629 1724 068 036After cross-foster 1563 1594 041 070At day 7 2699 2777 072 059At day 14 4377 4606 100 026At day 21 6117 6651 142 006

Piglet mean BW kgAt birth 144 156 009 004After cross-foster 157 163 003 030At day 7 270 284 005 018At day 14 457 478 007 012At day 21 649 694 009 001

Piglet ADG gdWeek 1 16141 17253 414 018Week 2 26412 27653 410 013Week 3 27312 30651 590 lt001Days 1ndash21 23361 25236 351 lt001

SEM standard error of means BW body weight ADG average daily gain1Dietary treatments C = control diet OEO = 15mgkg oregano essential oilsdiet

Moreover the OEO diet increased the counts of sowsrsquofaecal Lactobacillus but decreased the counts of Enterococcusand Escherichia coli Actually it has been demonstratedthat dietary supplementation with essential oils containingcarvacrol and thymol decreases populations of Escherichiacoli in broiler chickens [30] and increases the proportionsof Lactobacillus in cecum of broilers [31] Lactobacillus hasthe ability to inhibit ROS production through fermentationof colon digesta and to inhibit the growth of Enterococcusfaecalis and Escherichia coli [32] The results of the presentstudy indicated that sows fed the OEO diet shifted microbialecology in favor of reducing ROS production that alleviatedoxidative stress and oxidative damage of sows The period oftransition between late pregnancy and lactation representsan enormous metabolic challenge to the high-yielding sowAlleviating oxidative stress could definitively benefit sowrsquoshealth status

We also found that supplementing the sow diet withOEO tended to increase lactation feed intake of sows Thisobservation is consistent with the work of Allan and Bilkei[16] During pregnancy and lactation the sow undergoes


COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)

Figure 3 Diet effects on the value of HOMA-IR (a) and HOMA-IS (b) of sows Plasma glucose and insulin concentrations before feedingweremeasured the indirectmethods to evaluate insulin sensitivity by homeostasismodel assessment (HOMA)HOMA-IR= [(fasting insulinmIUL) times (fasting glucose mmolL)]225 HOMA-IS = 1[(fasting insulin mIUL) times (fasting glucose mmolL)] Values are means plusmn SEM(119899 = 5) ABEffect of sampling day (119875 lt 001) Effect of dietary treatment (119875 lt 01) lowastEffect of dietary treatment (119875 lt 005) There was atreatment times sampling day interaction for HOMA-IS value of sows (119875 lt 005) C = control diet OEO = 15mgkg oregano essential oils diet

numerous physiologic and metabolic changes such as pro-gressive and reversible insulin resistance corresponding toa decreased effectiveness of insulin to regulate blood glu-cose [33] Moreover insulin resistance during the peripartalperiod has negatively impacted the lactation feed intakeof sows [8 9] Sows fed the OEO diet tended to improvetheir insulin sensitivity during late gestation (HOMA valuesFigure 3) Excessive ROS has been shown to affect the insulinsignaling cascade and then the most common outcome ofdisrupted insulin signaling is insulin resistance [7] Thus wespeculated that supplementation of OEO in sow diets mayimprove insulin sensitivity during late pregnancy by affectingROS clearance in serum of sows

In the present trial sows fed the OEO diet exhib-ited significantly increased piglets ADG which can usuallyindicate an improvement of the amount andor qualityof colostrum and milk as they are major determinantsof litter performance [34] With regard to the quality ofcolostrum andmilk our results showed no differences amongthe dietary treatments in their nutrient compositions andimmunoglobulin concentration contradicting with previousfinding of Ariza-Nieto et al [17] who reported that OEOadministered to lactating sows reduced fat percentage inmilkon days 7 and 14 Discrepancies may be due to differencesbetween the duration of treatments (gestation and lactationversus lactation) and the dose (15mgkg versus 250mgkg)

Since no differences of the quality of colostrum and milkwere found the improvement of piglet performance can onlybe explained by the increase of their amount Actually we didfind a tendency of increased sowsrsquo lactation feed intake withthe supplementation diets which resulted in the production

of a higher amount of colostrum and milk [35 36] It wasunexpected to note that sows fed the OEO diet showed morebackfat thickness loss during lactation despite their increasedlactation feed intake This might be due to their higher litterweight which might have pushed them to use their bodyreserves for milk production

5 Conclusion

Our results demonstrated that there is an increased systemicoxidative stress during late gestation and early lactation ofsows The OEO supplementation to sowsrsquo diet during ges-tation and lactation improved performance of their pigletswhich may be attributed to the reduced oxidative stress

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by Fundamental Research Funds fortheCentral Universities (2013 PY047 nos 2012MBDX010 and2011PY019) and the Science and Technology Plan Researchand Development Projects of Hubei Province in the TwelfthFive-year Plan Period (no YJN0135) The authors thankMeriden Animal Health Ltd for providing oregano essentialoilTheywould like to thankYangXiang Joint StockCompanyfor providing sow feeding facilities


References

[1] A Agarwal K Makker and R Sharma ldquoClinical relevance ofoxidative stress in male factor infertility an updaterdquo AmericanJournal of Reproductive Immunology vol 59 no 1 pp 2ndash112008

[2] C B Berchieri-Ronchi S W Kim Y Zhao C R Correa K-JYeum and A L A Ferreira ldquoOxidative stress status of highlyprolific sows during gestation and lactationrdquo Animal vol 5 no11 pp 1774ndash1779 2011

[3] J Lapointe ldquoMitochondria as promising targets for nutritionalinterventions aiming to improve performance and longevity ofsowsrdquo Journal of Animal Physiology and Animal Nutrition vol98 no 5 pp 809ndash821 2014

[4] Y Zhao W L Flowers A Saraiva K-J Yeum and S WKim ldquoEffect of social ranks and gestation housing systems onoxidative stress status reproductive performance and immunestatus of sows1rdquo Journal of Animal Science vol 91 no 12 pp5848ndash5858 2013

[5] Y Zhao W L Flowers A Saraiva K-J Yeum and S W KimldquoEffect of heat stress on oxidative stress status and reproductiveperformance of sowsrdquo Journal of Animal Science vol 40 article108 2011

[6] RVinayagamoorthi Z Bobby andMG Sridhar ldquoAntioxidantspreserve redox balance and inhibit c-Jun-N-terminal kinasepathway while improving insulin signaling in fat-fed ratsevidence for the role of oxidative stress on IRS-1 serine phos-phorylation and insulin resistancerdquo Journal of Endocrinologyvol 197 no 2 pp 287ndash296 2008

[7] J L Rains and S K Jain ldquoOxidative stress insulin signaling anddiabetesrdquo Free Radical Biology and Medicine vol 50 no 5 pp567ndash575 2011

[8] E Mosnier N le Flocrsquoh M Etienne P Ramaekers B Seve andM-C Pere ldquoReduced feed intake of lactating primiparous sowsis associated with increased insulin resistance during the peri-partum period and is not modified through supplementationwith dietary tryptophanrdquo Journal of Animal Science vol 88 no2 pp 612ndash625 2010

[9] W C Weldon A J Lewis G F Louis J L Kovar MA Giesemann and P S Miller ldquoPostpartum hypophagia inprimiparous sows I Effects of gestation feeding level on feedintake feeding behavior and plasma metabolite concentrationsduring lactationrdquo Journal of Animal Science vol 72 no 2 pp387ndash394 1994

[10] A Sivropoulou E Papanikolaou C Nikolaou S KokkiniT Lanaras and M Arsenakis ldquoAntimicrobial and cytotoxicactivities ofOriganum essential oilsrdquo Journal of Agricultural andFood Chemistry vol 44 no 5 pp 1202ndash1205 1996

[11] S Burt ldquoEssential oils their antibacterial properties and poten-tial applications in foodsmdasha reviewrdquo International Journal ofFood Microbiology vol 94 no 3 pp 223ndash253 2004

[12] R J W Lambert P N Skandamis P J Coote and G-J ENychas ldquoA study of the minimum inhibitory concentration andmode of action of oregano essential oil thymol and carvacrolrdquoJournal of AppliedMicrobiology vol 91 no 3 pp 453ndash462 2001

[13] R Amorati M C Foti and L Valgimigli ldquoAntioxidant activityof essential oilsrdquo Journal of Agricultural and FoodChemistry vol61 no 46 pp 10835ndash10847 2013

[14] I Berniakovich M Trinei M Stendardo et al ldquop66119878ℎ119888-generated oxidative signal promotes fat accumulationrdquo Journalof Biological Chemistry vol 283 no 49 pp 34283ndash34293 2008

[15] C Mauch and G Bilkei ldquoStrategic application of oregano feedsupplements reduces sow mortality and improves reproductiveperformancemdasha case studyrdquo Journal of Veterinary Pharmacol-ogy andTherapeutics vol 27 no 1 pp 61ndash63 2004

[16] P Allan and G Bilkei ldquoOregano improves reproductive perfor-mance of sowsrdquoTheriogenology vol 63 no 3 pp 716ndash721 2005

[17] CAriza-NietoM Bandrick S K Baidoo L Anil TWMolitorand M R Hathaway ldquoEffect of dietary supplementation oforegano essential oils to sows on colostrum and milk com-position growth pattern and immune status of suckling pigsrdquoJournal of Animal Science vol 89 no 4 pp 1079ndash1089 2011

[18] J Lykkesfeldt and O Svendsen ldquoOxidants and antioxidants indisease oxidative stress in farm animalsrdquo Veterinary Journalvol 173 no 3 pp 502ndash511 2007

[19] P Y Van G J Hamilton I V Kremenevskiy et al ldquoLyophilizedplasma reconstituted with ascorbic acid suppresses inflamma-tion and oxidative DNA damagerdquo Journal of TraumamdashInjuryInfection and Critical Care vol 71 no 1 pp 20ndash25 2011

[20] F L Hou R F Zhang M W Zhang et al ldquoHepatoprotectiveand antioxidant activity of anthocyanins in black rice bran oncarbon tetrachloride-induced liver injury in micerdquo Journal ofFunctional Foods vol 5 no 4 pp 1705ndash1713 2013

[21] V Pialoux P J Hanly G E Foster et al ldquoEffects of exposure tointermittent hypoxia on oxidative stress and acute hypoxic ven-tilatory response in humansrdquo American Journal of Respiratoryand Critical Care Medicine vol 180 no 10 pp 1002ndash1009 2009

[22] D Du Y-H Shi and G-W Le ldquoThe effect of diet with differentglycemic index on the redox status of duodenums in miceand its underlying mechanismrdquo European Food Research andTechnology vol 230 no 6 pp 935ndash941 2010

[23] TMWallace J C Levy and D RMatthews ldquoUse and abuse ofHOMA modelingrdquo Diabetes Care vol 27 no 6 pp 1487ndash14952004

[24] M Saker N Soulimane Mokhtari S A Merzouk H MerzoukB Belarbi and M Narce ldquoOxidant and antioxidant statusin mothers and their newborns according to birthweightrdquoEuropean Journal of Obstetrics Gynecology and ReproductiveBiology vol 141 no 2 pp 95ndash99 2008

[25] S Arikan D Konukoglu C Arikan T Akcay and I DavasldquoLipid peroxidation and antioxidant status inmaternal and cordbloodrdquoGynecologic and Obstetric Investigation vol 51 no 3 pp145ndash149 2001

[26] F Inanc M Kilinc G Kiran et al ldquoRelationship betweenoxidative stress in cord blood and route of deliveryrdquo FetalDiagnosis and Therapy vol 20 no 5 pp 450ndash453 2005

[27] Z K Zeng S Zhang H L Wang and X S Piao ldquoEssential oiland aromatic plants as feed additives in non-ruminant nutri-tion a reviewrdquo Journal of Animal Science and Biotechnology vol6 article 7 10 pages 2015

[28] A R Abdallah A M Mansour and A A EL-Arabey ldquoThy-mol and carvacrol prevent cisplatin-induced nephrotoxicity byabrogation of oxidative stress inflammation and apoptosis inratsrdquo Journal of Biochemical and Molecular Toxicology vol 29no 14 pp 165ndash172 2015

[29] M Llana-Ruiz-Cabello D Gutierrez-Praena M PuertoS Pichardo A Jos and A M Camean ldquoIn vitro pro-oxidantantioxidant role of carvacrol thymol and their mixturein the intestinal Caco-2 cell linerdquo Toxicology in Vitro vol 29no 4 pp 647ndash656 2015

[30] A Roofchaee M Irani M A Ebrahimzadeh andM R AkbarildquoEffect of dietary oregano (Origanum vulgare L) essential oil on


growth performance cecal microflora and serum antioxidantactivity of broiler chickensrdquo African Journal of Biotechnologyvol 10 no 32 pp 6177ndash6183 2011

[31] D Jamroz A Wiliczkiewicz T Wertelecki J Orda and JSkorupinska ldquoUse of active substances of plant origin in chickendiets based on maize and locally grown cerealsrdquo British PoultryScience vol 46 no 4 pp 485ndash493 2005

[32] J Sun X-L Hu G-W Le and Y-H Shi ldquoLactobacilli preventhydroxy radical production and inhibit Escherichia coli andEnterococcus growth in systemmimicking colon fermentationrdquoLetters in AppliedMicrobiology vol 50 no 3 pp 264ndash269 2010

[33] M-C Pere and M Etienne ldquoInsulin sensitivity during preg-nancy lactation and postweaning in primiparous giltsrdquo Journalof Animal Science vol 85 no 1 pp 101ndash110 2007

[34] A Kingrsquoori ldquoSow lactation colostrum and milk yield a reviewrdquoJournal of Animal Science Advances vol 2 no 6 pp 525ndash5332012

[35] J Y Dourmad J Noblet and M Etienne ldquoEffect of proteinand lysine supply on performance nitrogen balance and bodycomposition changes of sows during lactationrdquo Journal ofAnimal Science vol 76 no 2 pp 542ndash550 1998

[36] A Ramanau H Kluge J Spilke and K Eder ldquoSupplementa-tion of sows with L-carnitine during pregnancy and lactationimproves growth of the piglets during the suckling periodthrough increased milk productionrdquo Journal of Nutrition vol134 no 1 pp 86ndash92 2004

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


objective of this study was to examine the effects of dietarysupplementation of sow diets with OEO during gestationand lactation on oxidative stress status colostrum and milkcomposition lactation feed intake and piglet performance

2 Materials and Methods

All experimental procedures were approved by the AnimalCare and Use Committee of Huazhong Agricultural Univer-sity

21 Animals Diet Treatments and Management Sixty largewhite sowswere originally allotted to the study the sowsweredivided into two groups (control group andOEOgroup) of 30animals After breeding six sows (4 in control group and 2 inOEO group) were returned to estrus within the estrus cycleFifty-four large white sows and parities of 495 plusmn 112 wereused in this trial After breeding the sows were randomlyallotted to 1 of 2 experimental dietary treatments based onparity and body weight (BW) control sows were fed a basaldiet (Table 1) with no supplementation (C 119899 = 26) and thetreatment sows were fed a basal diet added with 15mgkgOEO through gestation and lactation (OEO 119899 = 28)The commercially available OEO is a mixture powder thatcontains 5 OEO of Origanum vulgare subsp hirtum plantsand 95 natural feed grade inert carrier For each kilogramof sow feed we supplemented 300mg Orego-Stim (MeridenAnimalHealth Ltd UK) that is 15mgOEOThe componentsof OEO were shown in Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552015525218 TheOEO supplement contained carvacrol (8192) and thy-mol (350) Sows from the two groups were restrict-fedwith their respective diets during gestation Sows were fed20 kgday from days 1 to 30 of gestation 25 kgday from days31 to 90 of gestation and 30 kgday from day 91 of gestationto farrowing The diets were supplied twice a day (0700 and1430) During lactation the diet was supplied three times aday (0700 1100 and 1730) to ensure sows ad libitum accessto feed Pregnant sows were housed individually in gestationstalls (22m times 07m times 11m) Sows were moved from thegestation stalls to the farrowing rooms on day 107 plusmn 2 ofgestation and then kept in individual farrowing crates withstalls (22m times 07m) in pens that provided space on bothsides of the stall (22m times 05m) for the pigs after birthBoth sows and piglets had free access to water Piglets werenot offered creep feed Sow milk was the only feed availableto the piglets during lactation During the experimentalperiod data from sows with illness serious lameness deathand reproductive failure were not included in the analyses(Table 2)

22 Performance Measurement BW and backfat thicknessof sows were measured on days 0 and 107 of pregnancywithin 24 h of farrowing and at weaning Backfat thicknessat 65mm on each side of the dorsal midline at the last rib(1198752) was measured using ultrasound (PIGLOG105 SFAK-

Technology) At farrowing the numbers of total pigletsborn and piglets born alive were recorded The piglets werecross-fostered within dietary treatment groups by 48 h after

Table 1 Composition of the gestation and lactation diets (as-fedbasis)

Item Gestation LactationIngredient Corn 5630 5440Soybean meal 43 CP 1000 2600Wheat bran 3040 1100Calcium carbonate 120 151Dicalcium phosphate 104 123Salt 040 026Mildewcide1 012 010Choline chloride 014 100Premix2 040 150

Nutrient compositionNet energy MJkg3 932 1036Crude protein 1406 1892Lysine 3 061 103Calcium 3 077 106Available phosphorus 3 033 045

1Mildewcide ammonium propionate2Provided per kg of diet Cu 30mg Fe 160mg Zn 160mg Mn 55mg I05mg Se 05 Co 08mg Cr 02mg Vitamin A 14000 IU Vitamin D32900 IU Vitamin E 120mg Vitamin K3 6mg Vitamin B1 24mg VitaminB2 85mg Vitamin B6 45mg Vitamin B12 003mg Vitamin H 055mgPantothenic acid 30mg Folic acid 5mg Nicotinamide 50mg3Calculated chemical concentrations using values for feed ingredients fromthe National Research Council (1998)

Table 2 The number of sows during the experimental periods

Item C1 OEO1

Breeding 26 28Culled during gestation2 3 1Parturition 23 27Culled during lactation2 1 2Weaning 22 251Dietary treatments C = control diet OEO = 15mgkg oregano essential oilsdiet2Data of sows that were ill seriously lame died during the study and hadreproductive failure were not included

farrowing to adjust the litter size The number of piglets persow ranged from 9 to 12 piglets At weaning the numbers ofweaned piglets were recorded Piglets were weighed within24 h of birth (day 1) and on days 7 14 and 21 The daily feedintake of sows during lactation was recorded each morningby weighing daily feed refusals

23 Samples Collection At 2 h after feeding on days 10 6090 and 109 of gestation and on days 1 3 7 and 21 of lactationbefore feeding on days 10 and 109 of gestation and on days 3and 7 of lactation blood samples were collected from sows (5sows per diet group with the similar parity) by ear vein witha minimum amount of stress into heparinized tubes (5mL)or in tubes containing no anticoagulant (5mL) Fasting sowswere selected for blood sampling after an overnight fastingperiod of 16 h during gestation and 12 h during lactation


















data analysis




2O2catalyzed by





3 Results







Gestation Lactation

COEO

0

200

400

600G

SH-P

x (U

mL)

G 109G 90G 60 L 21L 1 G 10 L 3 L 7

(a)

COEO

lowast

C C B AB A B C C

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

1

2

3

4

TBA

RS (n

mol

mL)

(b)

COEO

C B AB AB AB A

lowast

AB AB

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

10

20

30

40

50

8-O

HdG

(ng

mL)

(c)

COEO

E E B B A C DE CD

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

2000

4000

6000

ROS

(RLU

)

(d)


4 Discussion





E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es


















COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















data analysis




2O2catalyzed by





3 Results







Gestation Lactation

COEO

0

200

400

600G

SH-P

x (U

mL)

G 109G 90G 60 L 21L 1 G 10 L 3 L 7

(a)

COEO

lowast

C C B AB A B C C

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

1

2

3

4

TBA

RS (n

mol

mL)

(b)

COEO

C B AB AB AB A

lowast

AB AB

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

10

20

30

40

50

8-O

HdG

(ng

mL)

(c)

COEO

E E B B A C DE CD

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

2000

4000

6000

ROS

(RLU

)

(d)


4 Discussion





E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es


















COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



2O2catalyzed by





3 Results







Gestation Lactation

COEO

0

200

400

600G

SH-P

x (U

mL)

G 109G 90G 60 L 21L 1 G 10 L 3 L 7

(a)

COEO

lowast

C C B AB A B C C

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

1

2

3

4

TBA

RS (n

mol

mL)

(b)

COEO

C B AB AB AB A

lowast

AB AB

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

10

20

30

40

50

8-O

HdG

(ng

mL)

(c)

COEO

E E B B A C DE CD

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

2000

4000

6000

ROS

(RLU

)

(d)


4 Discussion





E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es


















COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Gestation Lactation

COEO

0

200

400

600G

SH-P

x (U

mL)

G 109G 90G 60 L 21L 1 G 10 L 3 L 7

(a)

COEO

lowast

C C B AB A B C C

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

1

2

3

4

TBA

RS (n

mol

mL)

(b)

COEO

C B AB AB AB A

lowast

AB AB

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

10

20

30

40

50

8-O

HdG

(ng

mL)

(c)

COEO

E E B B A C DE CD

Gestation Lactation

G 109G 90G 60 L 21L 1 G 10 L 3 L 70

2000

4000

6000

ROS

(RLU

)

(d)


4 Discussion





E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es


















COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


E coli Entero Lacto

COEO

lowastlowast

lowast

0

5

10

15

Log 1

0CF

Ug

faec

es


















COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


COEO

Gestation Lactation

B B A B3

4

5

6

7H

OM

A-IR

G 109 L 3 L 7G 10

(a)

COEO

A A B A

Gestation Lactation

G 109 L 3 L 7G 100006

0008

0010

0012

0014

HO

MA-

IS

(b)






5 Conclusion




Acknowledgments



References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article effects of dietary supplementation of oregano...

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