The Effects of Feeding Fish Oil on Uterine Secretion of PGF2α, Milk Composition, and Metabolic ...

12 Pages·2004·0.11 MB·English

J. Dairy Sci. 87:921–932
 American Dairy Science Association, 2004.
The Effects of Feeding Fish Oil on Uterine Secretion of PGF , Milk
2α
Composition, and Metabolic Status of Periparturient Holstein Cows
R. Mattos,1 C. R. Staples,1 A. Arteche,1 M. C. Wiltbank,2 F. J. Diaz,2
T. C. Jenkins,3 and W. W. Thatcher1
1DepartmentofAnimalSciences,UniversityofFlorida,
Gainesville32611-0920
2DepartmentofDairyScience,UniversityofWisconsin,
Madison53706
3DepartmentofAnimal,Dairy,andVeterinarySciences,
ClemsonUniversity,Clemson,SC29634
ABSTRACT (Key words: fish oil, prostaglandin, uterus, n-3 fatty
acid)
Theobjectivesweretodeterminetheeffectofdietary
fishoil(FO)onuterinesecretionofPGF ,milkproduc- Abbreviation key: CLA = conjugated linoleic acid,
2α
tion,milkcomposition,andmetabolicstatusduringthe DA = displaced abomasum, DHA = docosahexaenoic
periparturientperiod.Holsteincowswereassignedran- acid, EPA = eicosapentaenoic acid, FO = fish oil,
domly to diets containing FO (n = 13) or olive oil (OO, G3PDH=glyceraldehyde3-phosphatedehydrogenase,
n=13).Cowswerefedprepartumandpostpartumdiets OO = olive oil, PGHS-2 = prostaglandin H synthase
2
that provided approximately 200 g/d from 21 d before 2, PGFM = 13, 14 dihydro, 15-keto PGF , PUN =
2α
the expected parturition until 21 d after parturition. plasma urea nitrogen.
The FO used contained 36% eicosapentaenoic acid
(EPA,C20:5,n-3)and28%docosahexaenoicacid(DHA,
INTRODUCTION
C22:6, n-3). Blood samples were obtained from 14 d
beforetheduedateuntild 21postpartum.Atotalof6 Fishoil(FO)containsrelativelyhighconcentrations
FO and 8 OO cows without periparturient disorders of two polyunsaturated fatty acids of the n-3 family:
wereusedinthestatisticalanalysesofPGF -metabo- eicosapentaenoic acid (EPA, C20:5) and docosahexae-
2α
lite (PGFM) and metabolite concentrations. Length of noic acid (DHA, C22:6). These fatty acids can be sup-
prepartumfeedingwithOOorFOdidnotdiffer.Propor- pliedonlybythedietbecauseEPAandDHAcannotbe
tions of individual and total n-3 fatty acids were in- synthesizeddenovoinmammaliansystems.Eicosapen-
creased in caruncular tissue and milk of cows fed FO. taenoicacidandDHAhaveinhibitedsecretionofPGF
2α
The combined concentrations of EPA and DHA in ca- indifferent animalcell culturesystems (Achardet al.,
runcular tissue were correlated positively with the 1997),includingbovineendometrialcells(Mattosetal.,
number of days supplemented with FO. Cows fed FO 2001). Inhibiting uterine secretion of PGF in vivo by
2α
had reduced concentrations of plasma PGFM during feeding EPA and DHA may reduce endometrial secre-
the 60 h immediately after parturition compared with tionofPGF topossiblyinduceanantiluteolyticeffect
2α
cows fed OO. Concentrations of prostaglandin H syn- duringearlypregnancyandincreasefertilityrates(Sta-
thase-2 mRNA and protein in caruncular tissue were ples et al., 1998; Mattos et al., 2000). Several studies
unaffected by diet. Production of milk and FCM were documented the effects of dietary marine products on
similar between cows fed the two oil diets. However, PGF secretion. Feeding fish meal, which contained
2α
cowsfedFOproducedlessmilkfat.FeedingFOreduced approximately8%FO(DMbasis),tolactatingmultipa-
plasma concentrations of glucose. Dietary fatty acids rouscowsfor47to51dreducedthesecretionofPGF
2α
given during the periparturient period can reduce the induced by estradiol and oxytocin injected on d 15 of
uterine secretion of PGF in lactating dairy cows and a synchronized estrous cycle (Mattos et al., 2002). In
2α
alter the fatty acid profile of milk fat. humans, consumption of large amounts of FO was re-
portedtoprolonggestation(Olsenetal.,1992),possibly
duetosuppressedsecretionofPGF delayingparturi-
2α
tion. Also, infusing ewes with 3 mL/kg of BW per day
ReceivedDecember19,2002. (i.v.) of an emulsion of FO containing 30% EPA and
AcceptedAugust8,2003.
20%DHAblockedabetamethasone-inducedincreasein
Corresponding author: William W. Thatcher; e-mail: thatcher@
animal.ufl.edu. plasmaconcentrationsofprostaglandinE2anddelayed
921
922 MATTOSETAL.
Table1.Numberofexperimentalcowsandincidenceofhealthdisorders.
Oliveoil Fishoil Total
Animalsstartingtheexperiment
Primiparous,n 5 4 9
Multiparous,n 8 9 17
Totalanimals,n 13 13 26
Animalsusedinstatisticalanalyses
Primiparous,n 1 2 3
Multiparous,n 7 4 11
Totalanimals,n 8 6 14
Healthdisorders
Dystocia,% 46(6/13) 38(5/13) 42(11/26)
Stillborncalf,% 8(1/13) 15(2/13) 12(3/26)
Retainedfetalmembranes,% 15(2/13) 15(2/13) 15(4/26)
Toxicmetritis,% 15(2/13) 8(1/13) 12(3/26)
Displacedabomasum,% 23(3/13) 38(5/13) 31(8/26)
occurrenceofparturitioninsheep(Baguma-Nibasheka date until parturition. It was then replaced by rations
et al., 1999). containingeitherFOorOO,whichwerefeduntilcows
A very intense secretion of uterine PGF , as evi- reached21dpostpartum.Duringtheprepartumperiod,
2α
denced by dramatic increases in concentration of cows were housed in sod-based pens and were moved
plasmaPGF ,ischaracteristicoftheimmediatepost- toafree-stallbarnatcalving.Cows(n=6)andheifers
2α
partum period in the cow (Guilbault et al., 1984a). (n=6)thathadmoderatetoseveredystocia,thatwere
Plasmaconcentrationsof13,14dihydro,15-ketoPGF diagnosed with displaced abomasum (DA), that re-
2α
(PGFM), the metabolite of PGF used to estimate tained fetal membranes, or that had toxic metritis
2α
PGF secretion, peaked at concentrations >1 ng/mL within 10 d after parturition were removed from the
2α
within1to3dafterparturitionanddecreasedprogres- analysesoftheeffectsofdietonplasmaconcentrations
sively until d 15 postpartum (Guibault et al., 1984a, ofPGFM,PUN,glucose,BHBA,andNEFA.Therefore,
1985). This period of intense secretion of uterine atotalofthreeheifers(1OO,2FO)and11cows(7OO,
PGF2αwasusedinthisexperimenttotestinhibitorsof 4 FO) were used in the statistical analyses because
prostanoid synthesis. their health status likely did not interfere with the
OurhypothesiswasthatfeedingEPAandDHAusing normal secretion of uterine PGF (Table 1).
2α
dietaryFOwouldincreasetheproportionofthesefatty Rations were formulated to provide approximately
acids in the uterine tissue of dairy cows and reduce 200 g/d of oil. Assumed average DMI were 10.2 and
thespontaneoussecretionofuterinePGF2αduringthe 11.6kg/dfortheclose-upandearlypostpartumperiods,
periparturientperiod.Ourobjectivesweretodetermine respectively. Because of anticipated lower DMI in the
the effect of dietary FO on spontaneous secretion of close-upperiod,nonlactatingcowrationscontained2%
PGF2α by the uterus during the periparturient period oil(DMbasis),whereaslactatingcowrationswerefor-
and to evaluate the effect of diet on milk production, mulatedtocontain1.8%oil.TheFOselectedcontained
milk composition, and indicators of metabolic status 36%EPAand28%DHA(Table2).Therefore,thecom-
such as plasma concentrations of glucose, NEFA, binedintakeofEPA(72g)andDHA(56g)wasexpected
BHBA, and plasma urea nitrogen (PUN). In addition, to be 128 g/d. Fish oil was fed at 1.8% of dietary DM
theeffectof dietonfattyacid compositionandconcen- postpartum in this study in order to deliver approxi-
trationsofprostaglandinH2synthase-2(PGHS-2)pro- mately50to60g/dofEPAplusDHA.Thiswasassum-
teinandmRNAincarunculartissueweredetermined. ing a ruminal escape of biohydrogenation of 65% and
an intestinal absorption of 70%.
MATERIALS AND METHODS Ingredientandchemicalcompositionofthedietsare
describedinTables3and4.Atparturition,rationswere
Cows and Diets
adjustedtosupplynutrientsrequiredduringearlylac-
The study was conducted from January 31 to April tation.
7, 2001. Pregnant Holstein cows (n = 17) and heifers Theconcentrateportionsofthedietsweremixedand
(n=9)wereassignedrandomlytodietscontainingFO stored in metal bins of 1.8-tonne capacity. Oils were
(n=13)(AristaIndustries,Wilton,CT)oroliveoil(OO, premixed using ground corn as a carrier. Concentrate
n = 13) (Classico, Bertolli, Italy). A ration containing mixturesandforagesourcesweremixedinaweighing
theoilswassuppliedfrom21dbeforetheexpecteddue and mixing unit (American Calan, Inc.) and offered
JournalofDairyScienceVol.87,No.4,2004
REPRODUCTIVEANDPRODUCTIONRESPONSESTOFISHOIL 923
Table2.Fattyacidprofileoffishoilandoliveoil. twice daily at 0700 and 1230 h to allow 5 to 10% orts
Diet (as-fed basis). Orts from each diet were collected once
dailyandweighed.Dailyortweightsforeachdietwere
Fattyacid1 Oliveoil Fishoil
subtracted from the ration offered and divided by the
(g/100goffattyacids) numberofcowsfedtodeterminedailyaverageindivid-
C14 0.00 0.41
ual cow intakes. The DM concentration of silage was
C14:1 0.00 0.09
C15:0 0.00 0.26 monitoredonceweekly(55°Cfor48h)tomaintainthe
C16:0 16.62 7.03 proper forage-to-concentrate ratio of diets. Samples of
C16:1 1.86 6.33
forages and concentrate mixes were collected weekly,
C18:0 2.70 1.00
tC18:1 0.00 1.44 compositedmonthly, andanalyzed forCP, NDF,ADF,
C18:1,n9 61.28 4.46 ether extract, and minerals (Dairy One, DHIA Forage
C18:2,n6 16.47 2.48
Testing Laboratory, Ithaca, NY) using wet chemistry.
CLAt9t11 0.00 0.71
CLAc9t11 0.00 0.00 Cows were milked 3× d at 0500, 1200, and 1900 h.
C18:3,n3 0.61 1.95 Calibrated electronic milk meters were used at each
C20:0 0.46 0.56
milking to record milk weights. Body condition scores
C21:0 0.00 6.36
C20:5,n3 0.00 35.81 and BW were determined at the introduction of diets,
C22:0 0.00 0.24 calving, and at d 21 postpartum.
C22:1 0.00 2.36
C22:6,n3 0.00 28.42
C24:0 0.00 0.09 Collection of Blood Samples
1Expressedasnumberofcarbons:doublebonds.
Blood(10mL)wasobtainedoncedailyat1730hfrom
14 d before due date until parturition and from d 15
until d 21 postpartum.Between the day of parturition
(d0)andd14postpartum,bloodsampleswerecollected
Table 3. Ingredient composition of experimental diets containing 2× per day at 0800 and 1730 h. Blood was collected
olive oil or fish oil fed to Holsteincows during late pregnancy and
from the coccygeal vessels into evacuated blood tubes
earlylactation.
containingEDTAasananticoagulant(10.5mg,Mono-
Feedstuff Nonlactating Lactating
ject, Sherwood Medical, St. Louis, MO). Samples were
%ofdietaryDM maintained in ice until plasma was separated by cen-
Cornsilage 37.6 28.0 trifugation (2600 × g, 30 min) at 4°C within 1 h of
Alfalfahay 6.3 11.9
Bermudahay 6.4 — collection and stored at −20°C until analyzed.
Cottonseedhulls — 4.8
Groundcorn 23.4 22.4
Soybeanmeal 9.2 14.1 PGFM and Metabolite Assays
Citruspulp 6.9 7.8
Oil 2.0 1.8 Samples collected between d 5 prepartum and d 21
Wholecottonseed 1.0 1.9 postpartumwereanalyzedforconcentrationsofPGFM
Pro-Lak1 1.2 2.2
Mineralmix2 5.5 — using a modification of the radioimmunoassay proce-
Mineralmix3 — 4.7 dure described by Mitchell et al. (1976). The PGFM
Tracemineralizedsalt4 0.5 — standard solutions were made by serial dilutions in a
Feedphos5 — 0.3
bufferofastocksolution(1µg/mLin10%ethanoland
1A marine and animal protein supplement containing 68% RUP 90%PBSbuffer)ofauthenticPGFM(Sigma,St.Louis,
(H.J.Baker&Bro.,Inc.,Stamford,CT).
MO).Standards(100µL)wereruninduplicatesatthe
2Mineralandvitaminmixcontaining22.8%CP,2.1%fat,22.89%
Ca,0.16%P,2.77%Mg,0.75%Na,0.20%K,2.42%S,8.03%Cl,146.7 followingconcentrations:15.6,31.2,62.5,125,250,500,
mg/kgofMn,95.0mg/kgofZn,26.6mg/kgofFe,112.5mg/kgofCu, 1000,2000,4000,and8000pg/mL.Thestandardcurve
10.7mg/kgofCo,7.9mg/kgofI,6.9mg/kgofSe,268,130IU/kgof
included 100 µL of prostaglandin-free plasma, which
vitaminA,40,000IU/kgofvitaminD,and1129IU/kgofvitaminE
(DMbasis). wasobtainedfromacyclingnonlactatingbeefcowpre-
3Mineralandvitaminmixcontaining26.4%CP,1.74%fat,10.15% treated twice with flunixin meglumine (Banamine,
Ca,0.90%P,3.1%Mg,8.6%Na,5.1%K,1.5%S,4.1%Cl,2231mg/ Schering-Plough,1g/i.m.injection),aPGHSinhibitor.
kgofMn,1698mg/kgofZn,339mg/kgofFe,512mg/kgofCu,31
Injections were given 16 h apart. Blood was collected
mg/kgofCo,26mg/kgofI,7.9mg/kgofSe,147,756IU/kgofvitamin
A,43,750IU/kgofvitaminD,and787IU/kgofvitaminE(DMbasis). 4hafterthesecondinjection,andtheplasmawassepa-
4Minimumconcentrationsof40%Na,55%Cl,0.25%Mn,0.2%Fe, ratedbycentrifugation.Samplesofprostaglandin-free
0.033%Cu,0.007%I,0.005%Zn,and0.0025%Co(DMbasis)(Flint plasma had undetectable concentrations (<10 pg/mL)
RiverMills,Inc.,Bainbridge,GA).
of PGFM when assayed utilizing standard curves in
5Contains21%Pand15to18%Ca(SoutheasternMinerals,Inc.,
Bainbridge,GA). 0.05 M PBS buffer. The PBS buffer contained 2.3 g/L
JournalofDairyScienceVol.87,No.4,2004
924 MATTOSETAL.
Table4.ChemicalcompositionofexperimentaldietscontainingeitheroliveorfishoilfedtoHolsteincows
duringlatepregnancyandearlylactation.
Oliveoil Fishoil Oliveoil Fishoil
Chemical nonlactating nonlactating lactating lactating
CP,%ofDM 14.03 13.96 16.7 17.20
NE ,Mcal/kgofDM1 1.68 1.68 1.73 1.74
L
NDF,%ofDM 31.17 30.85 30.49 28.40
ADF,%ofDM 19.39 19.11 19.43 18.75
Etherextract,%ofDM 5.32 4.77 5.28 5.36
NFC2 41.4 42.4 39.2 40.7
Ca,%ofDM 2.20 2.06 1.48 1.44
P,%ofDM 0.39 0.37 0.48 0.53
Mg,%ofDM 0.44 0.42 0.40 0.43
K,%ofDM 1.38 1.37 1.57 1.58
Na,%ofDM 0.26 0.27 0.41 0.48
S,%ofDM 0.27 0.28 0.22 0.24
Cl,%ofDM 1.22 1.18 0.42 0.42
Fe,ppmofDM 206 210 250 242
Zn,ppmofDM 65 64 112 114
Mn,ppmofDM 51 49 88 114
Cu,ppmofDM 16 12 25 38
1CalculatedbyDHIForageTestingLaboratory,Ithaca,NY.
2Calculatedas(%NFC=100%−%CP− %NDF−%fat−%ash).
NaH PO H O, 4.76 g/L Na HPO 7H O, 1 g/L sodium inhibition curve (heterogeneity of regression, P > 0.1).
2 4 2 2 4 2
azide, and 8.41 g/L NaCl. The buffer pH was adjusted Inter-andintraassaycoefficients ofvariationwere9.8
to 7.5 with NaOH. and 11.9%, respectively.
Activity and volume of radioactively labeled PGFM Plasma concentrations of NEFA and BHBA were
(Amersham Pharmacia Biotech, Piscataway, NJ) used measured in samples collected on d −5, −3, −1, 1, 4, 7,
were 18,000 dpm and 100 µL, respectively. For un- 10,13,16,and19,relativetothedayofparturition.A
knownsamples,finalassayvolumewas400µL;itcom- kitassaywasusedtomeasureplasmaconcentrationsof
prised100µLofsample,100µLofrabbitantiserumto BHBA(BHBA,kit310-A,SigmaDiagnostics,St.Louis,
PGFM(1:10,000;Meyer etal.,1995),100 µLofbuffer, MO). Inter- and intraassay coefficients of variation
and 100 µL of labeled PGFM. After a 12-h incubation were 7.3 and 17.6%, respectively. Plasma concentra-
at 4°C, free PGFM was separated using 750 µL of a tionsofNEFAweremeasuredwiththeWakoNEFAC
solution of charcoal-coated dextran (1.25% dextran test kit (Wako Chemicals USA, Richmond, VA). Inter-
[Sigma, St. Louis, MO], 12.5% activated charcoal and intraassay coefficients of variation were 2.9 and
[Sigma,St.Louis,MO]inaPBSbuffer).Aftercentrifu- 5.4%, respectively.
gation for 20 min at 3565 × g, the supernatant was Concentrationsof plasmaglucose andurea nitrogen
transferredtoscintillationvialsandmixedwith4.5mL weredetermineddaily fromd−5tod 21withanauto-
of scintillation fluid (Scintiverse II, Fisher Scientific, matedcolorimetricprocedure,whichutilizedanautoa-
Pittsburgh, PA). Activity was measured using a liquid nalyzer(AutoAnalyzerII,Bran+Luebbe,BuffaloGrove,
scintillation counter (model LKB 1219, Wallac Inc., IL).Theglucoseprocedureisamodification(Technicon
Gaithersburg, MD). Accuracy of the assay was deter- Industrial Method #339-19, Bran+Luebbe, Buffalo
mined by determining known quantities of exogenous Grove, IL) of that described by Gochman and Schmitz
PGFM previously added to prostaglandin-free cow (1972). The procedure for determination of urea nitro-
plasma.Addedmassesachievedfinalconcentrationsof gen(IndustrialMethodUS-339-01,Bran+Luebbe,Buf-
125, 250, 500, and 1000 pg/mL. Recovery of added (X) faloGrove,IL)isamodificationofthecarbamido-diace-
versus measured (Y) PGFM concentrations was de- tyl reaction procedure (Coulombe and Favreau, 1963).
scribedbylinearregression(Y= 1.1957×−56.72,R2=
0.98). Sensitivity of the assay was established at 15.6
Milk Fat Isolation and Analysis
pg/mL,becauseanantibodydilutionof1:10000andan
assay volume of 100 µL of plasma permitted detection Milk samples for the determination of fat, protein,
ofaminimummassof1.56pg.Dilutioninprostaglan- and somatic cells (Southeast Milk Inc., Belleview, FL)
din-freeplasmaofahigh(826.6pg/mL)PGFMplasma were collected weekly from two consecutive milkings.
sampleto100,50,and25%oforiginalvolumeresulted Milk fat and protein concentrations were analyzed by
inaninhibitioncurvethatwasparalleltothestandard the mid-infrared spectroscopic method. Milk samples
JournalofDairyScienceVol.87,No.4,2004
REPRODUCTIVEANDPRODUCTIONRESPONSESTOFISHOIL 925
also were collected during 2 consecutive milkings on Isolation of mRNA from Placentomes
the last experimental day (d 21) for analysis of fatty
Total RNA was isolated using the RNAgents total
acids using GLC. Samples for fatty acid analysis were
RNAisolationsystem(Promega,Madison,WI).Placen-
refrigerated at 4°C and subsequently composited ac-
tomesweregroundbrieflyinamortarandpestlecooled
cordingtothemilkyieldrecordedforeachofthemilk-
with liquid nitrogen. Approximately 30 mg of tissue
ings.Milkfatwasextractedusingadetergentsolution
was transferred to a fresh tube containing 900 µL of
containing 3% Triton X-100 (wt/vol) and 7% sodium
denaturing solution and homogenized for 20 s using a
hexametaphosphateindistilledwater.Asampleofmilk
polytrontissuegrinder.Next90µLof2M sodiumace-
fatwasmethylatedin0.5Msodiummethoxideinmeth-
tateand900µLofasolutioncontainingphenol,chloro-
anol followed by a second methylation in acetyl chlo-
form, and indole-3-acetic acid was added to the lysate
ride:methanol (1:10, vol/vol) as described by Kramer
and incubated on ice for 15 min. Samples were centri-
et al. (1997). Fatty acid methyl esters in milk were
fugedfor20minat14,000×ginarefrigeratedmicrocen-
separatedbyGLC(HP5890,AgilentTechnologies,Palo
trifuge. The supernatant was transferred to a fresh
Alto,CA)ona30m×0.25mm×0.2µm-filmthickness
tube and RNA was precipitated with an equal volume
SP2380capillarycolumn(Supleco,Inc.,Bellefonte,PA).
ofisopropanolandincubatedat−20°Cfor1h.Samples
Thecolumnovenwasprogrammedtorise4°C/minfrom
werethencentrifugedat14,000×gfor10mintopellet
aninitialtemperatureof50°C(heldfor2min)toafinal
RNA and washed with 1 ml of 70% ethanol. The RNA
temperatureof250°C(heldfor15min).Temperatures
pelletwasdriedandresuspendedin30µLofdiethylene
of the injector (100:1 split) and detector (flame ioniza-
pyrocarbonate-treated water. The RNA purity and
tion)ovenswere250°and260°C,respectively.Helium
quantity was measured by absorbance at 260/280 nm
was the carrier gas (20 cm/s).
in a spectrophotometer.
Efficiency of transfer of EPA and DHA from diet to
milkwascalculatedbydividingthemeanmassoffatty
acidsecretedinmilkdaily(averagedailymilkproduc- Determination of PGHS-2 mRNA
tion×averagemilkfatpercentage×averageproportion Concentrations in Placentomes
of EPA or DHA in milk fat) by the mean mass of that
Evaluation of PGHS-2 mRNA was accomplished
fatty acid consumed daily with the diet (average daily
using glyceraldehyde-3-phosphate dehydrogenase
DMI × estimated concentration of EPA or DHA in the
(G3PDH) mRNA as an internal control. Primers for
diet). Milk fat and fish oil were corrected for glycerol,
G3PDH were designed from the published sequences
assuming 10% by weight.
and yielded the expected 106-base pair product. The
PGHS-2 primers yielded the expected 484-base pair
Collection of Caruncles fragment and were designed as described previously
(Tsaietal.,1996).Reversetranscriptionwascarriedout
Caruncles were collected from 23 cows by manual with 19 µL of 1× master mix (1× reverse transcription
extraction through the vagina within 12 h of parturi-
buffer, 0.2 mM deoxynucleotides, 100 pmol random
tion.Tominimizebacterialcontaminationoftherepro-
primer,and40Uofreversetranscriptase)and1µLof
ductivetract,theperinealareawaswashedwithadis- sample for 1.5 h at 37°C. For PCR, 4 µL of reverse
infectant solution and dried with a paper towel. The transcriptionreactionwasaddedto1×PCRmastermix
technician used a shoulder-length sleeve and sterile (1×thermophilicbuffersuppliedwithenzyme,1.5mM
lubrication. After collection, caruncles were washed
MgCl , 0.2 mM deoxynucleotides, 0.4 µM each of for-
2
with water, plunged in liquid nitrogen, and stored at
ward and reverse primer pairs, and 1 U of Taq DNA
−80°C. Subsamples of frozen caruncles were subse- polymerase) for 28 cycles (95°C for 30 s, 57°C for 30 s,
quently used for total RNA extraction, preparation of and72°Cfor30s,followedbyafinalextensionat72°C
tissue lysates for protein analyses, and determination
for 5 min). Reaction products were separated on a 5%
offattyacidcomposition.Approximately7gofcaruncu-
PAGEgelandstainedwithethidiumbromide.Foreach
lartissuewerefreeze-driedfor24husingalyophilizer
sample,PGHS-2andG3PDHproductswerequantified
(Labconco, Kansas City, MO) to obtain a final mass of
usingtheCollageimagingsystem(FotodyneHartland,
approximately1.5gofDM.Fattyacidsindrycaruncu-
WI; Tsai et al., 1996).
lartissueweremethylatedandseparatedbygaschro-
matograph using the same techniques described for
Determination of PGHS-2 Protein
milk fatty acid composition. The only change was the
Concentrations in Placentomes
temperatureprogramofthegaschromatograph,which
increased from 140°C (3 min) to 220°C (20 min) at The PGHS-2 protein was analyzed by immunoblot.
3.7°C/min. Frozenuterinetissue(~60mg)washomogenizedin400
JournalofDairyScienceVol.87,No.4,2004
926 MATTOSETAL.
µLofcoldhomogenizationbuffer(50mMTris[pH7.4], within treatment × parity, and second-order interac-
150 mM NaCl, 1 mM sodium orthovanadate, 1 mM tions between treatment, parity, and time.
phenylmethylsulfonyl fluoride, 1 mg/mL leupeptin, 1 Dataonmilkfattyacidprofilesandsteady-statelev-
mg/mL aprotinin, 1% Triton X-100, and 0.25% deoxy- elsofPGHS-2mRNAandproteinincarunculartissue
cholate) using a polytron tissue grinder. Lysate was were analyzed using the GLM procedure. The model
centrifuged twice at 16,000 × g for 10 min to obtain a foranalysisofmilkfattyacidprofiles,PGHS-2mRNA,
clear lysate.An equal volumeof 2× loadingbuffer was and PGHS-2protein included the effectsof treatment.
added, and samples were steamed for 5 min. Samples The steady-state levels of G3PDH mRNA and β-actin
(20 to 30 µL) and 6.25 to 100 ng of PGHS-2 standards protein were included as a covariate in the model for
(Cayman Chemical, Ann Arbor, MI) were loaded on a analyses of PGHS-2 mRNA and PGHS-2 protein, re-
10%SDS-PAGEgelandproteinswereseparatedat120 spectively, to adjust for loading. Effects of treatment
mA for 1.5 h. Proteins were transferred to polyvinyli- onthenumberofdaysinfeedprepartumandthediffer-
dene fluoride membrane using the mini-protean II gel enceindaysbetweentheexpectedduedateandactual
transfer system (Bio-Rad, Hercules, CA). Following calvingdatealsoweretestedusingtheGLMprocedure.
transfer, blots were incubated in blocking buffer (10 Themodelincludedtheeffectoftreatmentonly.Dataon
mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and differenceswereconsideredsignificantatthe5%level.
5%NDM)overnightat4°C.Immunoblottingproceeded
by incubating blots with antiPGHS-2 antibody (Cay-
RESULTS AND DISCUSSION
man Chemical, Ann Arbor, MI) at 1:2000 dilution for
2 h at 25°C, followed by 3× washing (10 mM Tris, pH ThelengthofprepartumfeedingwithOO(22.5±2.8)
7.4,150mMNaCl,and0.1%Tween20).TheantiPGHS- or FO (21.8 ± 3.3) did not differ (P > 0.05). The range
2antibodydoesnotcross-reactwithPGHS-1.Antirab- ofdaysfedintheprepartumperiodwas10to39.Length
bithorseradishperoxidase(SantaCruzBiotechnology, of feeding did not affect the PGFM response of experi-
Santa Cruz, CA) was added at 1:5000 dilution for 1 h mental cows (P > 0.05). Concentrations of EPA and
at25°C,followedby3×washing.Specificproteinswere DHA in caruncular tissue were increased in cows fed
detected with enhanced chemiluminescent reagent FO (P < 0.01), indicating that the dietary fatty acids
(NEN Life Science Products, Boston, MA). Blots were were incorporated into uterine tissue (Table 5). The
exposed to X-ray film for 1 min and quantified using combined concentrations of caruncular EPA and DHA
the Collage imaging system. The PGHS-2 standards werecorrelatedpositivelywiththenumberofdayssup-
were used to generate a standard curve (Y = 2859X + plementedwithFO(r2=0.64,P<0.01),suggestingthat
24969, R2 = 0.99) to determine the amount of PGHS-2 introducingFObefored21beforetheexpectedduedate
presentintheuterinesamples.Toverifyequalprotein couldhavefurtherincreasedtheconcentrationsofEPA
loading, membranes were stripped and reprobed with andDHAintheuterus.Resultsfromotherstudiessup-
anti-actin primary antibody (Sigma, St. Louis, MO) at port the concept that this period of feeding was suffi-
1:20,000 dilution and antimouse-Horseradish Peroxi- cient to allow incorporation of dietary EPA and DHA
dase (Santa Cruz Biotechnology, Santa Cruz, CA) at intomembranephospholipidsoftheuterinecaruncles.
1:5000 following the same procedure as for PGHS-2. Intravenous infusion with n-3 fatty acids reduced
plasma concentrations of prostaglandin E after 4 d
2
in sheep (Baguma-Nibasheka et al., 1999). In another
Statistical Analyses
study, feeding rats with a diet rich in n-3 fatty acids
Plasma concentrations of PGFM, glucose, NEFA, for 3 wk resulted in a 50% replacement of the uterine
BHBA, and PUN were analyzed using the repeated phospholipidpoolofn-6fattyacidswithn-3fattyacids.
measuresanalysisofthemixedprocedureofSAS(Lit- Thereversewastruewhenthesupplyofn-3fattyacids
telletal.,1996).Covariancestructurestestedincluded waslimited.Changesinuterinefattyacidcomposition
compound symmetry, autoregressive 1, and unstruc- weredetected3wkaftertheintroductionoftreatment
tured. The model included effects of treatment, cow diets (Howie et al., 1992).
within treatment, time, and treatment × time interac- Cows fed FO had reduced concentrations of plasma
tion. Differences among treatment means of the treat- PGFM during the period of maximum secretion in the
ment×dayinteractionweretestedusingthesliceoption early postpartum period compared with cows fed OO.
of the mixed procedure. Differences weresignificant (P< 0.05) at0, 0.5, 2,and
Milkand4%FCMproduction,percentagesoffatand 2.5dpostpartum(Figure1).Thepatternofpostpartum
proteininmilk,andyieldsofproteinandfatwereana- concentrations of plasma PGFM was similar to what
lyzed using the mixed procedure of SAS. The model was previously reported. The inhibitory effects of FO
included the effects of treatment, parity, time, cow on concentrations of plasma PGFM are in agreement
JournalofDairyScienceVol.87,No.4,2004
REPRODUCTIVEANDPRODUCTIONRESPONSESTOFISHOIL 927
Table 5. Fatty acid profile of caruncles of Holstein cows fed diets containing olive or fish oil (% of total
fattyacidintissue).
Diet
Fattyacid1 Oliveoil Fishoil SE Treatment
(g/100goffattyacids)
C14 1.30 1.24 0.10 0.69
C14:1 0.27 0.18 0.05 0.25
C15:0 0.41 0.47 0.06 0.50
C16:0 19.84 21.79 0.74 0.09
C16:1 1.11 1.10 0.11 0.91
C18:0 24.97 25.53 0.47 0.04
tC18:1 1.09 2.44 0.21 <0.01
C18:1,n9 19.92 20.18 0.77 0.81
C18:2,n6 12.22 9.30 0.46 <0.01
CLAt9t11 0.35 0.27 0.07 0.40
CLAc9t11 0.36 0.35 0.05 0.88
C18:3,n3 0.51 0.44 0.08 0.52
C20:0 1.10 0.89 0.05 <0.01
C20:5,n3 0.23 1.66 0.02 <0.01
C22:0 5.09 4.09 0.20 <0.01
C22:6,n3 0.59 3.11 0.37 <0.01
C24:0 0.70 0.63 0.08 0.53
1Expressedasnumberofcarbons:doublebonds.
withthestudyofThatcheretal.(1997),whichdemon- concentration of plasma PGFM between parturition
strated that feeding diets containing 5.4% fish meal and d 5 tended to be significant (r2 = −0.14, P < 0.09).
(DM basis) attenuated the plasma PGFM response to A sustained difference in plasma PGFM concentra-
injections of estradiol-17β and oxytocin given on d 15 tions between cows fed OO and FO was not observed
of the estrous cycle of dairy cows. Mattos et al. (2002) throughout the experimental period. Plasma PGFM
described similar effects of increasing concentrations concentrations of cows fed OO and FO converged at
ofdietaryfishmeal(0,2.6,5.2,and7.8%ofdietaryDM) approximately d 5 and remained similar until the end
onsecretionofuterinePGF2αinducedbyestradioland oftheexperiment.TheearlyreductioninplasmaPGFM
oxytocin on d 15 of the estrous cycle. In the present concentrations could be explained by an FO-induced
study, the increased concentrations of inhibitory EPA reductioninPGF syntheticactivityincarunculartis-
2α
and DHA in caruncular tissue of cows fed FO suggest sue, which is a major source of PGF (Guilbault et
2α
that these fatty acids may be the active components al., 1984a, 1984b). With the normal early shedding of
reducing secretion of PGF2α. The correlation between caruncular tissue (Gier and Marion, 1968) in both
caruncularconcentrationsofEPAandDHAandmean groups, the subsequent residual uterine production of
PGF may not have been sufficient to detect possible
2α
differences attributed to the diets.
Diet did not affect the number of days between the
expectedduedateandactualcalvingdate.Itwasantici-
patedthatreduceduterinePGF secretioncouldresult
2α
indelayedparturition.CowsfedOOandFOcalved3.4
± 1.8 and 3.3 ± 2.1 d before the due date, respectively
(P>0.05).However,correlationanalysisindicatedthat
late calving was associated with increasing concentra-
tions of EPA and DHA in caruncular tissue (r2 = 0.52,
P < 0.01). Although it is tempting to speculate that
increased EPA and DHA concentration in caruncular
tissue cause delayed parturition, there is not a clear
cause-effect relationship between the two variables.
Late parturition due to causes unrelated to feeding
Figure1.Pre-andpostpartumplasmaconcentrationsofprosta- could result in increased concentrations of EPA and
glandinF metabolite(PGFM)ofcowsfedfishoil(▲,n=6)orolive
2α DHAduetoalongerfeedingperiodandyieldasignifi-
oil((cid:1),n=8)(LSM+SE).ThePGFMconcentrationswerelowerin
cowsfedfishoilat0,0.5,2,and2.5dafterparturition(*,P<0.05). cant correlation.
JournalofDairyScienceVol.87,No.4,2004
928 MATTOSETAL.
Table6.Dry matterintake,milkproductionand milkcompositionofHolsteincowsfed dietscontaining
oliveoilorfishoil.
Measurement Oliveoil Fishoil SE P
Primiparous,n 1 2
Multiparous,n 7 4
DMI,prepartumkg/d 13.5 9.4 3.21
DMI,postpartumkg/d 12.7 10.4 4.01
Milk,kg/d 25.0 20.4 3.4 0.37
Milkfat,% 5.20 4.35 0.5 0.22
Milkprotein,% 3.51 3.27 0.2 0.49
4%FCM,kg/d 26.1 20.1 3.6 0.27
Milkfat,kg/d 1.27 0.81 0.13 0.03
Milkprotein,kg/d 0.86 0.63 0.11 0.18
1StandardDeviation.
Feeding FO attenuated the rise in plasma PGFM and FO cows, respectively (P = 0.19). This is in
concentrations that occurs in the postpartum period. agreement with the findings described by Mattos et
ThemechanismbywhichdietaryEPAandDHAinhibit al. (2001), which indicated that polyunsaturated fatty
secretion of uterine prostanoids likely requires their acidssuchasEPAandDHAdidnotaffectsteady-state
absorption from the intestinal lumen and subsequent levels of PGHS-2 mRNA in bovine endometrial cells.
incorporation into cellular lipid pools of the endome- Inaddition,concentrationsofPGHS-2proteinincarun-
trium.Inthisexperiment,absorptionofEPAandDHA culartissuewereunaffectedbydiet(16.8±5.3vs.16.4
from the diet was demonstrated by the increased con- ±5.5ng/mgtissueforcowsfedOOorFO,respectively,
centrationsofthesefattyacidsinmilkfat.Inaddition, P = 0.93).
supplemental FOincreased the concentrationsof EPA In addition to competitive and inhibitory mecha-
and DHA in caruncular tissue. Similarly, feeding fish nisms, a lower efficiency of conversion of EPA to
mealincreasedtheproportionofEPAandDHAinendo- prostanoids of the 3 series may result in reduced total
metriallipidsofbeefcows(Burnsetal.,2000),indicat- prostanoid synthesis. The PGHS-2 converts EPA into
ingthatdietarychangescanalterthefattyacidcompo- prostanoidsofthe3seriesinalesstime-efficientman-
sition of the uterus. ner than it converts arachidonic acid into prostanoids
Synthesis of PGF involves cleavage of fatty acids of the 2 series (Kulmacz et al., 1994).
2α
from plasma membrane phospholipids. These cleaved Daily DMI was reduced by 30.3 and 18.1% in the
fattyacids generallyare unsaturatedand includeara- prepartumandpostpartumperiods,respectively,when
chidonic acid and EPA, which can be converted to FOreplacedOOinthediet(Table6).Inpreviousstudies
prostanoidsofthe2and3series,respectively.Increased (AbuGhazalehetal.,2002;Whitlocketal.,2002),FOfed
availability of EPA in membrane phospholipids as a at2%ofdietaryDMwasdetrimentaltoDMI,whereasa
result of feeding FO could displace arachidonic acid, similar intake of oil from extruded soybeans was not
leading to increased synthesis of prostanoids of the 3 detrimental.
seriesattheexpenseofprostanoidsofthe2series,such Averagemilkproductionbetween0and21dpostpar-
asPGF .Prostanoidsofthe3seriesarelessbioactive tum was unaffected (P > 0.05) by diet although least
2α
(Needleman et al., 1979), and there appears to be no squaremeansweredifferentby4.6kg/d(Table6).These
evidence for their role in ruminant luteolysis. Prosta- results agree with those of AbuGhazaleh et al. (2002),
glandinF hasonly25%affinityfortheovinelutealFP Donovan et al. (2000), and Whitlock et al. (2002), who
3α
receptor comparedwith PGF (Balapure etal., 1989). also reported no effect of feeding FO at 2% of dietary
2α
Docosahexaenoicacidmayactinadifferentmanner DM.Milkproteinandfatconcentrationsalsowereunaf-
and reduce expression of the PGHS enzymes (Achard fected(P>0.05)bydiet(Table6).Othershavereported
et al., 1997), which could make these enzymes less no effect of feeding FO at 2% of dietary DM on milk
availableandfurtherreduceprostanoidsynthesis.Do- protein concentration, but milk fat concentration has
cosahexaenoic acid is not a substrate for the PGHS been reduced (Donovan et al., 2000; AbuGhazaleh et
enzymes,butitisastrongcompetitiveinhibitoroftheir al.,2002;Whitlocketal.,2002).Productionof4%FCM
activity (Corey et al., 1983). and milk protein was not affected, whereas milk fat
However,concentrationsofPGHS-2mRNAincarun- production was less by cows fed FO than by cows fed
clescollectedwithin12hofparturitionwereunaffected OO(P<0.05).Parity×treatmentinteractionswerenot
bydiet.Thesteady-statelevelsofPGHS-2were37080 significant for each of these measurements. Changes
± 6569 and 46597 ± 6094 densitometric units for OO inBW inthe prepartumand postpartumperiods were
JournalofDairyScienceVol.87,No.4,2004
REPRODUCTIVEANDPRODUCTIONRESPONSESTOFISHOIL 929
Table 7.Milk fattyacid profile ofHolstein cows feddiets containingolive oil orfish oil(% of totalfatty
acidinmilk).
Diet
Normal Treatment
Fattyacid1 Oliveoil Fishoil Milk2 SE P
(g/100goffattyacids)
C4:0 2.01 1.85 3.29 0.13 0.40
C6:0 1.45 1.25 1.80 0.21 0.52
C8:0 0.69 0.61 1.20 0.13 0.66
C10:0 1.37 1.22 2.40 0.26 0.69
C12:0 1.45 1.30 2.69 0.25 0.67
C14:0 6.25 5.51 10.18 0.72 0.48
C14:1 0.59 0.53 — 0.04 0.31
C16:0 25.95 25.07 26.35 0.70 0.40
C16:1 1.61 1.79 2.40 0.12 0.30
C18:0 14.88 11.38 11.98 0.95 0.03
C18:1,n9 27.41 28.99 25.15 4.05 0.79
C18:2,n6 1.42 0.16 2.40 0.57 0.15
CLAt9t11 0.011 0.100 — 0.01 <0.01
CLAc9t11 0.46 0.68 — 0.09 0.12
CLAt10c12 0.00 0.021 — 0.009 0.12
TotalCLA3 0.469 0.799 — 0.10 0.04
C18:3,n3 0.084 0.162 1.50 0.005 <0.01
C20:1,n9 0.343 0.346 — 0.012 0.87
C20:5,n3 0.003 0.422 — 0.06 <0.01
C22:6,n3 0.013 0.467 — 0.08 <0.01
Other 6.68 10.75 — 0.75 <0.01
Totaln-34 0.100 1.050 — 0.15 0.01
Totaln-65 1.422 0.163 — 0.57 0.15
Totaln-96 33.50 34.84 — 1.66 0.59
Saturated 55.45 49.92 — 1.57 0.03
Unsaturated 37.69 39.18 — 1.84 0.59
1Expressedasnumberofcarbons:doublebonds.
2NationalDairyCouncil,1993.
3TotalCLA:cis-9,trans-11C18:2+trans-9,trans-11C18:2+cis-10,trans-12C18:2.
4n-3fattyacids:C18:3+C20:5+C22:6.
5n-6fattyacids:C18:2.
6n-9fattyacids:trans-C18:1+cis-C18:1+C20:1+C22:1.
not affected by diet (P > 0.05), suggesting that energy FeedingFOincreasedtheconcentrationsoftotalcon-
status was similar between the two groups. jugatedlinoleicacids(CLA)(P<0.01).Increasedtotal
Feeding FO increased the proportion of individual CLAconcentrationsasaresultoffeedingn-3fattyacids
and total n-3 fatty acids (linolenic, EPA, and DHA) in alsohavebeenreported(Cantetal.,1997;Franklinet
milk when compared to cows fed OO (P < 0.01, Table al.,1999;Donovanetal.,2000).Anincreasedconversion
7). These results agree with previous reports (Cant et of dietary linoleic and linolenic acids to CLA caused
al., 1997; Donovan et al., 2000). The rates of transfer by FO may explain the higher concentrations of CLA
fromdiettomilkforEPAandDHAinthisstudywere observed (Whitlock et al., 2002).
5.4and7.6%,respectively.Transferefficiencyhasbeen Plasma concentrations of NEFA were not affected
reported to be greater for DHA (16.2%) than for EPA by diet (P > 0.05, Figure 2). Concentrations of NEFA
(9.3%) (Cant et al., 1997). The increase in concentra- increased during the week before parturition, sug-
tions of n-3 fatty acids in milk from cows fed FO oc- gesting increased fat mobilization and a decreasing
curredinconcertwithdecreasedconcentrationsofsatu- DMI. After parturition, NEFA concentrations de-
rated fatty acids (P = 0.03). This reduction is largely creasedslowlyfrom900to600µEq/Lbyd21postpar-
due to a reduction in concentrations of C18:0 in milk tum. This profile of periparturient NEFA concentra-
fat from cows fed FO. Similar effects of FO on total tionsissimilartothatreportedbyGarcia-Bojaliletal.
saturatedfattyacidsandonC18:0havebeenreported (1998).PlasmaconcentrationsofBHBAincreasedwith
(Franklinetal.,1999;Donovanetal.,2000).Thepropor- increasing days postpartum (P < 0.02, Figure 3). Con-
tion of total long-chain fatty acids (> 18 carbons) was centrations of BHBA tended to be greater in cows fed
not affected by diet (P > 0.05). FO(P=0.08,diet×dayinteraction).Pairwisecompari-
JournalofDairyScienceVol.87,No.4,2004
930 MATTOSETAL.
Figure2.Pre-andpostpartumplasmaconcentrationsofNEFA
of cows fed fish oil (▲, n = 6) or olive oil ((cid:1), n = 8) (LSM ± SE). Figure4.Profilesofplasmaglucoseconcentrationsofperipartur-
Concentrationswerenotaffectedbydiet(P>0.05). ientcowsfeddietscontainingfishoil(▲,n=6)oroliveoil((cid:1),n=
8)(LSM+SE).(**,P≤0.05;*P≤0.1).Themaineffectofthediet
issignificant(P=0.03).
sons of treatment means indicated differences on d 10
(P = 0.05) and 13 (P = 0.10). The timing of peak and
rangeofBHBAconcentrationsofthisstudyweresimi- gluconeogenesis and therefore reduced plasma glu-
lar to those reported by Vazquez-An˜on et al. (1997). cose concentrations.
FeedingFOreducedplasmaconcentrationsofglucose Concentrations of PUN were greater in the prepar-
(P=0.03,Figure4).Concentrationspeakedat1dbefore tumperiodincowsfedFO,asdeterminedbydifferences
parturitionand declinedprogressivelyuntild 13to15 detectedond−5andd−3(P<0.1,Figure5).Postpartum
postpartum.Duffieldetal.(1998)reportedsimilarpro- PUN concentrations were similar to those reported by
files of postpartum glucose concentrations. The effect Roseler et al. (1993) for rations supplying 100% of the
of FO on glucose concentrations could be due to the requirements of degradable intake protein and unde-
reduced DMIassociated with that diet.Another possi- gradedintakeproteinforlactatingdairycows(National
bleexplanationinvolvestheinhibitionofgluconeogenic Research Council, 2001).
enzymes by components of FO. Eicosapentaenoic acid The incidence of postpartum health disorders was
inhibited the expression of phosphoenolpyruvate car- unaffectedbydiet(Table1).TheincidenceofDA(31%,
boxykinaseinrathepatomacells(Murataetal.,2001). 8/26)wasconsiderablygreaterthanwhatisconsidered
This enzyme catalyzes the conversion of oxaloacetate normal for high producing herds. A survey of 61 high-
tophosphoenolpyruvate,whichisarequiredstepinthe
conversion of propionic acid to glucose. It is possible
that EPA supplied in the FO diet may have inhibited
Figure3.Pre-andpostpartumplasmaconcentrationsofBHBA Figure5.Profilesofplasmaureanitrogenofperiparturientcows
of cows fed fish oil (▲, n = 6) or olive oil ((cid:1), n = 8) (LSM ± SE). feddietscontainingfishoil(▲,n=6)oroliveoil((cid:1),n=8)(LSM+
ConcentrationsofBHBAweregreaterincowsfedfishoilat10and SE).SignificantdifferencesweredetectedatDay−5andDay−3.(**,
13dafterparturition(*,P<0.10;**,P=0.05). P≤0.05;*P≤0.1).
JournalofDairyScienceVol.87,No.4,2004