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The Effects of Feeding Fish Oil on Uterine Secretion of PGF₂, Milk Composition, and Metabolic Status of Periparturient Holstein Cows

¹ Department of Animal Sciences, University of Florida, Gainesville 32611-0920
² Department of Dairy Science, University of Wisconsin, Madison 53706
³ Department of Animal, Dairy, and Veterinary Sciences, Clemson University, Clemson, SC 29634

Corresponding author: William W. Thatcher; e-mail: thatcher{at}animal.ufl.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION IMPLICATIONS ACKNOWLEDGEMENTS REFERENCES

 
The objectives were to determine the effect of dietary fish oil (FO) on uterine secretion of PGF₂, milk production, milk composition, and metabolic status during the periparturient period. Holstein cows were assigned randomly to diets containing FO (n = 13) or olive oil (OO, n = 13). Cows were fed prepartum and postpartum diets that provided approximately 200 g/d from 21 d before the expected parturition until 21 d after parturition. The FO used contained 36% eicosapentaenoic acid (EPA, C20:5, n-3) and 28% docosahexaenoic acid (DHA, C22:6, n-3). Blood samples were obtained from 14 d before the due date until d 21 postpartum. A total of 6 FO and 8 OO cows without periparturient disorders were used in the statistical analyses of PGF₂-metabolite (PGFM) and metabolite concentrations. Length of prepartum feeding with OO or FO did not differ. Proportions of individual and total n-3 fatty acids were increased in caruncular tissue and milk of cows fed FO. The combined concentrations of EPA and DHA in caruncular tissue were correlated positively with the number of days supplemented with FO. Cows fed FO had reduced concentrations of plasma PGFM during the 60 h immediately after parturition compared with cows fed OO. Concentrations of prostaglandin H synthase-2 mRNA and protein in caruncular tissue were unaffected by diet. Production of milk and FCM were similar between cows fed the two oil diets. However, cows fed FO produced less milk fat. Feeding FO reduced plasma concentrations of glucose. Dietary fatty acids given during the periparturient period can reduce the uterine secretion of PGF₂ in lactating dairy cows and alter the fatty acid profile of milk fat.

Key Words: fish oil • prostaglandin • uterus • n-3 fatty acid

Abbreviation key: CLA = conjugated linoleic acid, DA = displaced abomasum, DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, FO = fish oil, G3PDH = glyceraldehyde 3-phosphate dehydrogenase, OO = olive oil, PGHS-2 = prostaglandin H₂ synthase 2, PGFM = 13, 14 dihydro, 15-keto PGF₂, PUN = plasma urea nitrogen

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION IMPLICATIONS ACKNOWLEDGEMENTS REFERENCES

 
Fish oil (FO) contains relatively high concentrations of two polyunsaturated fatty acids of the n-3 family: eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6). These fatty acids can be supplied only by the diet because EPA and DHA cannot be synthesized de novo in mammalian systems. Eicosapentaenoic acid and DHA have inhibited secretion of PGF₂ in different animal cell culture systems (Achard et al., 1997), including bovine endometrial cells (Mattos et al., 2001). Inhibiting uterine secretion of PGF₂ in vivo by feeding EPA and DHA may reduce endometrial secretion of PGF₂ to possibly induce an antiluteolytic effect during early pregnancy and increase fertility rates (Staples et al., 1998; Mattos et al., 2000). Several studies documented the effects of dietary marine products on PGF₂ secretion. Feeding fish meal, which contained approximately 8% FO (DM basis), to lactating multiparous cows for 47 to 51 d reduced the secretion of PGF₂ induced by estradiol and oxytocin injected on d 15 of a synchronized estrous cycle (Mattos et al., 2002). In humans, consumption of large amounts of FO was reported to prolong gestation (Olsen et al., 1992), possibly due to suppressed secretion of PGF₂ delaying parturition. Also, infusing ewes with 3 mL/kg of BW per day (i.v.) of an emulsion of FO containing 30% EPA and 20% DHA blocked a betamethasone-induced increase in plasma concentrations of prostaglandin E₂ and delayed occurrence of parturition in sheep (Baguma-Nibasheka et al., 1999).

A very intense secretion of uterine PGF₂, as evidenced by dramatic increases in concentration of plasma PGF₂, is characteristic of the immediate postpartum period in the cow (Guilbault et al., 1984a). Plasma concentrations of 13, 14 dihydro, 15-keto PGF₂ (PGFM), the metabolite of PGF₂ used to estimate PGF₂ secretion, peaked at concentrations >1 ng/mL within 1 to 3 d after parturition and decreased progressively until d 15 postpartum (Guibault et al., 1984a, 1985). This period of intense secretion of uterine PGF₂ was used in this experiment to test inhibitors of prostanoid synthesis.

Our hypothesis was that feeding EPA and DHA using dietary FO would increase the proportion of these fatty acids in the uterine tissue of dairy cows and reduce the spontaneous secretion of uterine PGF₂ during the periparturient period. Our objectives were to determine the effect of dietary FO on spontaneous secretion of PGF₂ by the uterus during the periparturient period and to evaluate the effect of diet on milk production, milk composition, and indicators of metabolic status such as plasma concentrations of glucose, NEFA, BHBA, and plasma urea nitrogen (PUN). In addition, the effect of diet on fatty acid composition and concentrations of prostaglandin H₂ synthase-2 (PGHS-2) protein and mRNA in caruncular tissue were determined.

	MATERIALS AND METHODS

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Cows and Diets
The study was conducted from January 31 to April 7, 2001. Pregnant Holstein cows (n = 17) and heifers (n = 9) were assigned randomly to diets containing FO (n = 13) (Arista Industries, Wilton, CT) or olive oil (OO, n = 13) (Classico, Bertolli, Italy). A ration containing the oils was supplied from 21 d before the expected due date until parturition. It was then replaced by rations containing either FO or OO, which were fed until cows reached 21 d postpartum. During the prepartum period, cows were housed in sod-based pens and were moved to a free-stall barn at calving. Cows (n = 6) and heifers (n = 6) that had moderate to severe dystocia, that were diagnosed with displaced abomasum (DA), that retained fetal membranes, or that had toxic metritis within 10 d after parturition were removed from the analyses of the effects of diet on plasma concentrations of PGFM, PUN, glucose, BHBA, and NEFA. Therefore, a total of three heifers (1 OO, 2 FO) and 11 cows (7 OO, 4 FO) were used in the statistical analyses because their health status likely did not interfere with the normal secretion of uterine PGF₂ (Table 1).

Cows were milked 3x d at 0500, 1200, and 1900 h. Calibrated electronic milk meters were used at each milking to record milk weights. Body condition scores and BW were determined at the introduction of diets, calving, and at d 21 postpartum.

Collection of Blood Samples
Blood (10 mL) was obtained once daily at 1730 h from 14 d before due date until parturition and from d 15 until d 21 postpartum. Between the day of parturition (d 0) and d 14 postpartum, blood samples were collected 2x per day at 0800 and 1730 h. Blood was collected from the coccygeal vessels into evacuated blood tubes containing EDTA as an anticoagulant (10.5 mg, Monoject, Sherwood Medical, St. Louis, MO). Samples were maintained in ice until plasma was separated by centrifugation (2600 x g, 30 min) at 4°C within 1 h of collection and stored at -20°C until analyzed.

PGFM and Metabolite Assays
Samples collected between d 5 prepartum and d 21 postpartum were analyzed for concentrations of PGFM using a modification of the radioimmunoassay procedure described by Mitchell et al. (1976). The PGFM standard solutions were made by serial dilutions in a buffer of a stock solution (1 µg/mL in 10% ethanol and 90% PBS buffer) of authentic PGFM (Sigma, St. Louis, MO). Standards (100 µL) were run in duplicates at the following concentrations: 15.6, 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 pg/mL. The standard curve included 100 µL of prostaglandin-free plasma, which was obtained from a cycling nonlactating beef cow pretreated twice with flunixin meglumine (Banamine, Schering-Plough, 1 g/i.m. injection), a PGHS inhibitor. Injections were given 16 h apart. Blood was collected 4 h after the second injection, and the plasma was separated by centrifugation. Samples of prostaglandin-free plasma had undetectable concentrations (<10 pg/mL) of PGFM when assayed utilizing standard curves in 0.05 M PBS buffer. The PBS buffer contained 2.3 g/L NaH₂PO₄ H₂O, 4.76 g/L Na₂HPO₄ 7H₂O, 1 g/L sodium azide, and 8.41 g/L NaCl. The buffer pH was adjusted to 7.5 with NaOH.

Activity and volume of radioactively labeled PGFM (Amersham Pharmacia Biotech, Piscataway, NJ) used were 18,000 dpm and 100 µL, respectively. For unknown samples, final assay volume was 400 µL; it comprised 100 µL of sample, 100 µL of rabbit antiserum to PGFM (1:10,000; Meyer et al., 1995), 100 µL of buffer, and 100 µL of labeled PGFM. After a 12-h incubation at 4°C, free PGFM was separated using 750 µL of a solution of charcoal-coated dextran (1.25% dextran [Sigma, St. Louis, MO], 12.5% activated charcoal [Sigma, St. Louis, MO] in a PBS buffer). After centrifugation for 20 min at 3565 x g, the supernatant was transferred to scintillation vials and mixed with 4.5 mL of scintillation fluid (Scintiverse II, Fisher Scientific, Pittsburgh, PA). Activity was measured using a liquid scintillation counter (model LKB 1219, Wallac Inc., Gaithersburg, MD). Accuracy of the assay was determined by determining known quantities of exogenous PGFM previously added to prostaglandin-free cow plasma. Added masses achieved final concentrations of 125, 250, 500, and 1000 pg/mL. Recovery of added (X) versus measured (Y) PGFM concentrations was described by linear regression (Y = 1.1957x - 56.72, R² = 0.98). Sensitivity of the assay was established at 15.6 pg/mL, because an antibody dilution of 1:10000 and an assay volume of 100 µL of plasma permitted detection of a minimum mass of 1.56 pg. Dilution in prostaglandin-free plasma of a high (826.6 pg/mL) PGFM plasma sample to 100, 50, and 25% of original volume resulted in an inhibition curve that was parallel to the standard inhibition curve (heterogeneity of regression, P > 0.1). Inter- and intraassay coefficients of variation were 9.8 and 11.9%, respectively.

Plasma concentrations of NEFA and BHBA were measured in samples collected on d -5, -3, -1, 1, 4, 7, 10, 13, 16, and 19, relative to the day of parturition. A kit assay was used to measure plasma concentrations of BHBA (BHBA, kit 310-A, Sigma Diagnostics, St. Louis, MO). Inter- and intraassay coefficients of variation were 7.3 and 17.6%, respectively. Plasma concentrations of NEFA were measured with the Wako NEFA C test kit (Wako Chemicals USA, Richmond, VA). Inter- and intraassay coefficients of variation were 2.9 and 5.4%, respectively.

Concentrations of plasma glucose and urea nitrogen were determined daily from d -5 to d 21 with an automated colorimetric procedure, which utilized an autoanalyzer (AutoAnalyzer II, Bran+Luebbe, Buffalo Grove, IL). The glucose procedure is a modification (Technicon Industrial Method #339-19, Bran+Luebbe, Buffalo Grove, IL) of that described by Gochman and Schmitz (1972). The procedure for determination of urea nitrogen (Industrial Method US-339-01, Bran+Luebbe, Buffalo Grove, IL) is a modification of the carbamido-diacetyl reaction procedure (Coulombe and Favreau, 1963).

Milk Fat Isolation and Analysis
Milk samples for the determination of fat, protein, and somatic cells (Southeast Milk Inc., Belleview, FL) were collected weekly from two consecutive milkings. Milk fat and protein concentrations were analyzed by the mid-infrared spectroscopic method. Milk samples also were collected during 2 consecutive milkings on the last experimental day (d 21) for analysis of fatty acids using GLC. Samples for fatty acid analysis were refrigerated at 4°C and subsequently composited according to the milk yield recorded for each of the milkings. Milk fat was extracted using a detergent solution containing 3% Triton X-100 (wt/vol) and 7% sodium hexametaphosphate in distilled water. A sample of milk fat was methylated in 0.5 M sodium methoxide in methanol followed by a second methylation in acetyl chloride:methanol (1:10, vol/vol) as described by Kramer et al. (1997). Fatty acid methyl esters in milk were separated by GLC (HP 5890, Agilent Technologies, Palo Alto, CA) on a 30 m x 0.25 mm x 0.2 µm-film thickness SP2380 capillary column (Supleco, Inc., Bellefonte, PA). The column oven was programmed to rise 4°C/min from an initial temperature of 50°C (held for 2 min) to a final temperature of 250°C (held for 15 min). Temperatures of the injector (100:1 split) and detector (flame ionization) ovens were 250° and 260°C, respectively. Helium was the carrier gas (20 cm/s).

Efficiency of transfer of EPA and DHA from diet to milk was calculated by dividing the mean mass of fatty acid secreted in milk daily (average daily milk production x average milk fat percentage x average proportion of EPA or DHA in milk fat) by the mean mass of that fatty acid consumed daily with the diet (average daily DMI x estimated concentration of EPA or DHA in the diet). Milk fat and fish oil were corrected for glycerol, assuming 10% by weight.

Collection of Caruncles
Caruncles were collected from 23 cows by manual extraction through the vagina within 12 h of parturition. To minimize bacterial contamination of the reproductive tract, the perineal area was washed with a disinfectant solution and dried with a paper towel. The technician used a shoulder-length sleeve and sterile lubrication. After collection, caruncles were washed with water, plunged in liquid nitrogen, and stored at -80°C. Subsamples of frozen caruncles were subsequently used for total RNA extraction, preparation of tissue lysates for protein analyses, and determination of fatty acid composition. Approximately 7 g of caruncular tissue were freeze-dried for 24 h using a lyophilizer (Labconco, Kansas City, MO) to obtain a final mass of approximately 1.5 g of DM. Fatty acids in dry caruncular tissue were methylated and separated by gas chromatograph using the same techniques described for milk fatty acid composition. The only change was the temperature program of the gas chromatograph, which increased from 140°C (3 min) to 220°C (20 min) at 3.7°C/min.

Isolation of mRNA from Placentomes
Total RNA was isolated using the RNAgents total RNA isolation system (Promega, Madison, WI). Placentomes were ground briefly in a mortar and pestle cooled with liquid nitrogen. Approximately 30 mg of tissue was transferred to a fresh tube containing 900 µL of denaturing solution and homogenized for 20 s using a polytron tissue grinder. Next 90 µL of 2 M sodium acetate and 900 µL of a solution containing phenol, chloroform, and indole-3-acetic acid was added to the lysate and incubated on ice for 15 min. Samples were centrifuged for 20 min at 14,000 x g in a refrigerated microcentrifuge. The supernatant was transferred to a fresh tube and RNA was precipitated with an equal volume of isopropanol and incubated at -20°C for 1 h. Samples were then centrifuged at 14,000 x g for 10 min to pellet RNA and washed with 1 ml of 70% ethanol. The RNA pellet was dried and resuspended in 30 µL of diethylene pyrocarbonate-treated water. The RNA purity and quantity was measured by absorbance at 260/280 nm in a spectrophotometer.

Determination of PGHS-2 mRNA Concentrations in Placentomes
Evaluation of PGHS-2 mRNA was accomplished using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA as an internal control. Primers for G3PDH were designed from the published sequences and yielded the expected 106-base pair product. The PGHS-2 primers yielded the expected 484-base pair fragment and were designed as described previously (Tsai et al., 1996). Reverse transcription was carried out with 19 µL of 1x master mix (1x reverse transcription buffer, 0.2 mM deoxynucleotides, 100 pmol random primer, and 40 U of reverse transcriptase) and 1 µL of sample for 1.5 h at 37°C. For PCR, 4 µL of reverse transcription reaction was added to 1x PCR master mix (1x thermophilic buffer supplied with enzyme, 1.5 mM MgCl₂, 0.2 mM deoxynucleotides, 0.4 µM each of forward and reverse primer pairs, and 1 U of Taq DNA polymerase) for 28 cycles (95°C for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min). Reaction products were separated on a 5% PAGE gel and stained with ethidium bromide. For each sample, PGHS-2 and G3PDH products were quantified using the Collage imaging system (Fotodyne Hartland, WI; Tsai et al., 1996).

Determination of PGHS-2 Protein Concentrations in Placentomes
The PGHS-2 protein was analyzed by immunoblot. Frozen uterine tissue (~60 mg) was homogenized in 400 µL of cold homogenization buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 1% Triton X-100, and 0.25% deoxycholate) using a polytron tissue grinder. Lysate was centrifuged twice at 16,000 x g for 10 min to obtain a clear lysate. An equal volume of 2x loading buffer was added, and samples were steamed for 5 min. Samples (20 to 30 µL) and 6.25 to 100 ng of PGHS-2 standards (Cayman Chemical, Ann Arbor, MI) were loaded on a 10% SDS-PAGE gel and proteins were separated at 120 mA for 1.5 h. Proteins were transferred to polyvinylidene fluoride membrane using the mini-protean II gel transfer system (Bio-Rad, Hercules, CA). Following transfer, blots were incubated in blocking buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% NDM) overnight at 4°C. Immunoblotting proceeded by incubating blots with antiPGHS-2 antibody (Cayman Chemical, Ann Arbor, MI) at 1:2000 dilution for 2 h at 25°C, followed by 3x washing (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). The antiPGHS-2 antibody does not cross-react with PGHS-1. Antirabbit horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) was added at 1:5000 dilution for 1 h at 25°C, followed by 3x washing. Specific proteins were detected with enhanced chemiluminescent reagent (NEN Life Science Products, Boston, MA). Blots were exposed to X-ray film for 1 min and quantified using the Collage imaging system. The PGHS-2 standards were used to generate a standard curve (Y = 2859X + 24969, R² = 0.99) to determine the amount of PGHS-2 present in the uterine samples. To verify equal protein loading, membranes were stripped and reprobed with anti-actin primary antibody (Sigma, St. Louis, MO) at 1:20,000 dilution and antimouse-Horseradish Peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:5000 following the same procedure as for PGHS-2.

Statistical Analyses
Plasma concentrations of PGFM, glucose, NEFA, BHBA, and PUN were analyzed using the repeated measures analysis of the mixed procedure of SAS (Littell et al., 1996). Covariance structures tested included compound symmetry, autoregressive 1, and unstructured. The model included effects of treatment, cow within treatment, time, and treatment x time interaction. Differences among treatment means of the treatment x day interaction were tested using the slice option of the mixed procedure.

Milk and 4% FCM production, percentages of fat and protein in milk, and yields of protein and fat were analyzed using the mixed procedure of SAS. The model included the effects of treatment, parity, time, cow within treatment x parity, and second-order interactions between treatment, parity, and time.

Data on milk fatty acid profiles and steady-state levels of PGHS-2 mRNA and protein in caruncular tissue were analyzed using the GLM procedure. The model for analysis of milk fatty acid profiles, PGHS-2 mRNA, and PGHS-2 protein included the effects of treatment. The steady-state levels of G3PDH mRNA and ß-actin protein were included as a covariate in the model for analyses of PGHS-2 mRNA and PGHS-2 protein, respectively, to adjust for loading. Effects of treatment on the number of days in feed prepartum and the difference in days between the expected due date and actual calving date also were tested using the GLM procedure. The model included the effect of treatment only. Data on differences were considered significant at the 5% level.

	RESULTS AND DISCUSSION

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The length of prepartum feeding with OO (22.5 ± 2.8) or FO (21.8 ± 3.3) did not differ (P > 0.05). The range of days fed in the prepartum period was 10 to 39. Length of feeding did not affect the PGFM response of experimental cows (P > 0.05). Concentrations of EPA and DHA in caruncular tissue were increased in cows fed FO (P < 0.01), indicating that the dietary fatty acids were incorporated into uterine tissue (Table 5). The combined concentrations of caruncular EPA and DHA were correlated positively with the number of days supplemented with FO (r² = 0.64, P < 0.01), suggesting that introducing FO before d 21 before the expected due date could have further increased the concentrations of EPA and DHA in the uterus. Results from other studies support the concept that this period of feeding was sufficient to allow incorporation of dietary EPA and DHA into membrane phospholipids of the uterine caruncles. Intravenous infusion with n-3 fatty acids reduced plasma concentrations of prostaglandin E₂ after 4 d in sheep (Baguma-Nibasheka et al., 1999). In another study, feeding rats with a diet rich in n-3 fatty acids for 3 wk resulted in a 50% replacement of the uterine phospholipid pool of n-6 fatty acids with n-3 fatty acids. The reverse was true when the supply of n-3 fatty acids was limited. Changes in uterine fatty acid composition were detected 3 wk after the introduction of treatment diets (Howie et al., 1992).

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View larger version (16K): [in this window] [in a new window]   Figure 1. Pre- and postpartum plasma concentrations of prostaglandin F2 metabolite (PGFM) of cows fed fish oil (, n = 6) or olive oil (, n = 8)(LSM + SE). The PGFM concentrations were lower in cows fed fish oil at 0, 0.5, 2, and 2.5 d after parturition (*, P < 0.05).

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Diet did not affect the number of days between the expected due date and actual calving date. It was anticipated that reduced uterine PGF₂ secretion could result in delayed parturition. Cows fed OO and FO calved 3.4 ± 1.8 and 3.3 ± 2.1 d before the due date, respectively (P > 0.05). However, correlation analysis indicated that late calving was associated with increasing concentrations 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 could result in increased concentrations of EPA and DHA due to a longer feeding period and yield a significant correlation.

Feeding FO attenuated the rise in plasma PGFM concentrations that occurs in the postpartum period. The mechanism by which dietary EPA and DHA inhibit secretion of uterine prostanoids likely requires their absorption from the intestinal lumen and subsequent incorporation into cellular lipid pools of the endometrium. In this experiment, absorption of EPA and DHA from the diet was demonstrated by the increased concentrations of these fatty acids in milk fat. In addition, supplemental FO increased the concentrations of EPA and DHA in caruncular tissue. Similarly, feeding fish meal increased the proportion of EPA and DHA in endometrial lipids of beef cows (Burns et al., 2000), indicating that dietary changes can alter the fatty acid composition of the uterus.

Synthesis of PGF₂ involves cleavage of fatty acids from plasma membrane phospholipids. These cleaved fatty acids generally are unsaturated and include arachidonic acid and EPA, which can be converted to prostanoids of the 2 and 3 series, respectively. Increased availability of EPA in membrane phospholipids as a result of feeding FO could displace arachidonic acid, leading to increased synthesis of prostanoids of the 3 series at the expense of prostanoids of the 2 series, such as PGF₂. Prostanoids of the 3 series are less bioactive (Needleman et al., 1979), and there appears to be no evidence for their role in ruminant luteolysis. Prostaglandin F₃ has only 25% affinity for the ovine luteal FP receptor compared with PGF₂ (Balapure et al., 1989).

Docosahexaenoic acid may act in a different manner and reduce expression of the PGHS enzymes (Achard et al., 1997), which could make these enzymes less available and further reduce prostanoid synthesis. Docosahexaenoic acid is not a substrate for the PGHS enzymes, but it is a strong competitive inhibitor of their activity (Corey et al., 1983).

However, concentrations of PGHS-2 mRNA in caruncles collected within 12 h of parturition were unaffected by diet. The steady-state levels of PGHS-2 were 37080 ± 6569 and 46597 ± 6094 densitometric units for OO and FO cows, respectively (P = 0.19). This is in agreement with the findings described by Mattos et al. (2001), which indicated that polyunsaturated fatty acids such as EPA and DHA did not affect steady-state levels of PGHS-2 mRNA in bovine endometrial cells. In addition, concentrations of PGHS-2 protein in caruncular tissue were unaffected by diet (16.8 ± 5.3 vs. 16.4 ± 5.5 ng/mg tissue for cows fed OO or FO, respectively, P = 0.93).

In addition to competitive and inhibitory mechanisms, a lower efficiency of conversion of EPA to prostanoids of the 3 series may result in reduced total prostanoid synthesis. The PGHS-2 converts EPA into prostanoids of the 3 series in a less time-efficient manner than it converts arachidonic acid into prostanoids of the 2 series (Kulmacz et al., 1994).

Daily DMI was reduced by 30.3 and 18.1% in the prepartum and postpartum periods, respectively, when FO replaced OO in the diet (Table 6). In previous studies (AbuGhazaleh et al., 2002; Whitlock et al., 2002), FO fed at 2% of dietary DM was detrimental to DMI, whereas a similar intake of oil from extruded soybeans was not detrimental.

Feeding FO increased the proportion of individual and total n-3 fatty acids (linolenic, EPA, and DHA) in milk when compared to cows fed OO (P < 0.01, Table 7). These results agree with previous reports (Cant et al., 1997; Donovan et al., 2000). The rates of transfer from diet to milk for EPA and DHA in this study were 5.4 and 7.6%, respectively. Transfer efficiency has been reported to be greater for DHA (16.2%) than for EPA (9.3%) (Cant et al., 1997). The increase in concentrations of n-3 fatty acids in milk from cows fed FO occurred in concert with decreased concentrations of saturated fatty acids (P = 0.03). This reduction is largely due to a reduction in concentrations of C18:0 in milk fat from cows fed FO. Similar effects of FO on total saturated fatty acids and on C18:0 have been reported (Franklin et al., 1999; Donovan et al., 2000). The proportion of total long-chain fatty acids (> 18 carbons) was not affected by diet (P > 0.05).

Plasma concentrations of NEFA were not affected by diet (P > 0.05, Figure 2). Concentrations of NEFA increased during the week before parturition, suggesting increased fat mobilization and a decreasing DMI. After parturition, NEFA concentrations decreased slowly from 900 to 600 µEq/L by d 21 postpartum. This profile of periparturient NEFA concentrations is similar to that reported by Garcia-Bojalil et al. (1998). Plasma concentrations of BHBA increased with increasing days postpartum (P < 0.02, Figure 3). Concentrations of BHBA tended to be greater in cows fed FO (P = 0.08, diet x day interaction). Pairwise comparisons of treatment means indicated differences on d 10 (P = 0.05) and 13 (P = 0.10). The timing of peak and range of BHBA concentrations of this study were similar to those reported by Vazquez-Añon et al. (1997).

View larger version (14K): [in this window] [in a new window]   Figure 2. Pre- and postpartum plasma concentrations of NEFA of cows fed fish oil (, n = 6) or olive oil (, n = 8) (LSM ± SE). Concentrations were not affected by diet (P > 0.05).

View larger version (13K): [in this window] [in a new window]   Figure 3. Pre- and postpartum plasma concentrations of BHBA of cows fed fish oil (, n = 6) or olive oil (, n = 8) (LSM ± SE). Concentrations of BHBA were greater in cows fed fish oil at 10 and 13 d after parturition (*, P < 0.10; **, P = 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 4. Profiles of plasma glucose concentrations of periparturient cows fed diets containing fish oil (, n = 6) or olive oil (, n = 8) (LSM + SE). (**, P 0.05; * P 0.1). The main effect of the diet is significant (P = 0.03).

View larger version (17K): [in this window] [in a new window]   Figure 5. Profiles of plasma urea nitrogen of periparturient cows fed diets containing fish oil (, n = 6) or olive oil (, n = 8) (LSM + SE). Significant differences were detected at Day -5 and Day -3. (**, P 0.05; * P 0.1).

	IMPLICATIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION IMPLICATIONS ACKNOWLEDGEMENTS REFERENCES

 
Uterine synthesis of PGF₂ may be reduced in dairy cows consuming elevated amounts of the n-3 fatty acids, EPA and DHA. If this same response initiated by select dietary fatty acids can be achieved around the time of pregnancy recognition, regression of the corpus luteum might be prevented and embryo survival enhanced in lactating dairy cows.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION IMPLICATIONS ACKNOWLEDGEMENTS REFERENCES

 
This research was supported in part by the NRI Competitive Grant Program/USDA, grant 98-35203-6367. This is Florida Agriculture Experimental Station Journal Series No. R-09849.

Received for publication December 19, 2002. Accepted for publication August 8, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION IMPLICATIONS ACKNOWLEDGEMENTS REFERENCES

 


AbuGhazaleh, A. A., D. J. Schingoethe, A. R. Hippen, K. F. Kalscheur, and L. A. Whitlock. 2002. Fatty acid profiles of milk and rumen digesta from cows fed fish oil, extruded soybeans, or their blend. J. Dairy Sci. 85:2266–2276.[Abstract/Free Full Text]

Achard, D., M. Gilbert, C. Benistant, S. B. Slama, D. L. DeWitt, W. L. Smith, and M. Lagarde. 1997. Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells. Biochem. Biophys. Res. Commum. 241:513–518.[Medline]

Baguma-Nibasheka, M., J. T. Brenna, and P. W. Nathanielsz. 1999. Delay of pre-term delivery in sheep by n-3 long chain polyunsaturates. Biol. Reprod. 60:698–701.[Abstract/Free Full Text]

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