J. Dairy Sci. 85:889–899 American Dairy Science Association, 2002.
Milk Production and Composition, Ovarian Function, and Prostaglandin Secretion of Dairy Cows Fed Omega-3 Fats1 H. V. Petit,*2 R. J. Dewhurst,† N. D. Scollan,† J. G. Proulx,‡ M. Khalid,3 W. Haresign,§ H. Twagiramungu,|| and G. E. Mann# *Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Lennoxville, QC, Canada J1M 1Z3 †Institute of Grassland and Environmental Research Plas Gogerddan, Aberystwyth, Wales SY23 3EB, UK ‡120 Chemin Lavalle´e, Katevale, QC, Canada J0B 1W0 §Institute of Rural Studies, The University of Wales of Aberystwyth Llanbadarn Campus, Aberystwyth, Wales SY23 3AL, UK ||L’Alliance Boviteq Inc. 1425 grand rang St-Franc¸ois, Saint-Hyacinthe, QC, Canada J2S 7A9 #Division of Animal Physiology, School of Biosciences, University of Nottingham Sutton Bonington, Loughborough LE12 5RD, UK
ABSTRACT Four multiparous Holstein cows were used in a 4 × 4 Latin square experiment to study the effects of fat sources rich in omega-3 fatty acids on milk production and composition, follicular development, and prostaglandin secretion. All cows were fed a total mixed diet containing 60% grass silage and 40% concentrate. The four treatments were concentrates based either on Megalac, formaldehyde-treated whole linseed, a mixture (50:50, oil basis) of fish oil and formaldehyde-treated whole linseed, or no fat source in the concentrate but with 500 g per day of linseed oil being infused into the duodenum. Feed intakes and milk yield were similar among treatments. In general, the lowest digestibility was observed for the formaldehyde-treated whole linseed treatment. Feeding fish oil decreased milk fat and protein percentages. Alpha-linolenic acid increased from 1.0 to 13.9% of milk fatty acids with linseed oil infusion. This confirms the high potential to incorporate α-linolenic acid into milk, and suggests that the formaldehyde treatment had little effect to limit biohydrogenation in the rumen. Increasing the supply of α-linolenic acid to these cows did not result in an increase in the concentration of eicosapentaenoic acid in milk. Levels of 13,14-dihydro-15-keto-PGF2α in plasma were higher
Received June 11, 2001. Accepted November 14, 2001. Corresponding author: H. V. Petit; e-mail:
[email protected]. 1 Contribution Number 725 from the Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, PO Box 90, Lennoxville, QC, Canada J1M 1Z3. 2 This study was conducted while H. V. Petit was on leave at the Institute of Grassland and Environmental Research. 3 Present address: Department of Farm Animal Medicine and Surgery, The Royal Veterinary College, Hawhshead Lane, North Mymms, Hatfield, HERTS, AL9 7TA, UK.
for cows receiving formaldehyde-treated linseed and fish oil. Increases in this metabolite in response to oxytocin challenge, tended to be lower for cows given linseed either as sole oil supplement in the diet or as a duodenal infusion of linseed oil. Follicle dynamics were similar among treatments. Larger corpora lutea (CL) were found with cows that received high levels of omega-3 fatty acids through the diet as formaldehydetreated linseed or as a mixture of formaldehyde-treated linseed and fish oil, although CL were smaller when cows were infused with linseed oil into the duodenum. These results suggest that the improvement in gestation rate that was observed when feeding increased levels of alpha-linolenic acid in earlier work may partly result from lower levels of production of the dienoic prostaglandin PGF2α. (Key words: dairy cow, linseed, fatty acids, ovarian function, prostaglandin) Abbreviation key: CL = corpora lutea; DHA = docosahexaenoic acid; EPA = eicosapentaenoic acid; FA = fatty acids; FIS = concentrate based on a mixture (50:50) of fish oil and formaldehyde-treated linseed; LCFA = longchain fatty acids; LIN = concentrate based on formaldehyde-treated linseed; MEG = concentrate based on Megalac; OIL = concentrate without fat but with 500 g of linseed oil infused daily in the duodenum; PG = prostaglandins; PGFM = 13,14-dihydro-15-ketoPGF2α; PUFA = polyunsaturated fatty acids. INTRODUCTION Milk yields of cows in the UK have increased dramatically as the proportion of Holstein genes has increased over the last 25 yr. However, this has coincided with a decline in the pregnancy rate to first service of 1% per year (Royal et al., 2000). Some of this alarming decline
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may relate directly to increased milk production and extended periods of negative energy balance in early lactation (Butler and Smith, 1989). However, there have also been significant changes in diets over this period, notably increased use of fat to increase the energy concentration in concentrates. Supplementary fats are likely to affect fertility because fatty acids (FA) are the precursors both of prostaglandins (PG) and, via cholesterol, the steroid hormones. Lucy et al. (1992) suggested that it was FA, not the additional energy provided by the FA, that stimulated ovarian function. There are two main families of essential FA, termed omega-3 and omega-6 FA, that could affect fertility. The main source of omega-6 FA is dietary linoleic acid (C18:2n-6), and this is converted to arachidonic acid (C20:4n-6), which inter alia is the precursor of the dienoic (2-series) PG, such as PGF2α. The same elongase and desaturase enzymes also convert the main dietary omega-3 FA (α-linolenic acid; C18:3n-3) to eicosapentaenoic acid (EPA; C20:5n-3), the precursor of the trienoic (3-series) PG, such as PGF3α (Abayasekara and Wathes, 1999). Competition between omega-3 and omega-6 precursors for desaturation and elongation, as well as at the site of PG synthetase, means that increasing the supply of omega-3 FA will decrease production of dienoic PG (Barnouin and Chassagne, 1991). In many cases the trienoic PG have lower biological activity than the corresponding dienoic PG (Fly and Johnston, 1990), and this may directly affect aspects of fertility. For example, treatments that reduce ovarian and endometrial synthesis of PGF2α, at the expense of PGF3α, may contribute to a reduction in embryonic mortality (Mattos et al., 2000). There is some evidence for different effects of α-linolenic acid and the omega-3 FA from fish oil (EPA and docosahexaenoic acid [DHA], C22:6n-3) on eicosanoid (interleukin) synthesis, perhaps because of differences in the way in which these FA incorporate into cell membranes (Wu et al., 1996). Supplementary fats can also reduce the total synthesis of PG by affecting the activity of PG synthase (Thatcher et al., 1995). There is growing interest in increasing levels of omega-3 FA in meat and milk because of benefits for human health (e.g., Burr, 1989). The present study was conducted with diets that were also used in studies of FA composition and flavor of beef (Choi et al., 2000). Linseed and fish oil were used as sources of omega-3 FA; linseed is very rich in α-linolenic acid (C18:3n-3), and fish oil is an excellent source of the n-3 FA, EPA, and DHA. The hypothesis of this experiment was that feeding a source rich in omega-3 FA would decrease 13,14-dihydro-15-keto-PGF2α (PGFM) secretion and affect follicular development. The effects of feeding omega-3 and omega-6 FA on feed intake, digestibility, Journal of Dairy Science Vol. 85, No. 4, 2002
Table 1. Chemical composition of grass silage.1 Item
Silage
pH DM, % ADF, % of DM NDF, % of DM Gross energy, Mcal/kg of DM Ether extract, % of DM CP, % of DM Soluble N, % of N NH3 N, % of N Lactic acid, % of DM Acetic acid, % of DM Propionic acid, % of DM Isobutyric acid, % of DM Butyric acid, % of DM Valeric acid, % of DM Fatty acids, % of total C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
3.70 29.5 25.1 42.8 5.01 4.19 20.2 28.2 10.6 9.3 1.4 0.04 0 0.01 0.01 0.24 0.52 18.4 0.21 0.15 0.30 16.5 63.7
1 Mean of 5-wk composite samples that were prepared from three weekly samples.
milk production, and composition were also determined. We also monitored plasma cholesterol levels because these can be affected by fat supplementation and they are the precursors for ovarian steroidogenesis (Carroll et al., 1990). MATERIALS AND METHODS Animals and Diets Four multiparous lactating Holstein cows with a mean of 158 DIM (SE = 7), and averaging 670 kg BW (SE = 47 kg), were used. The cows had previously been prepared with simple cannulae in the rumen and duodenum and were kept in individual stalls, bedded on rubber mats with sawdust, and with free access to water and mineral blocks. Cows were milked twice a day at 0730 and 1600 h. Silage (Table 1) was prepared from a single sward of perennial ryegrass without wilting, using a precision chop forage harvester (Alois Pottinger MBH Mex VI PU457, Grieskirchen, Germany) set to a theoretical chop length of 5 cm. Add-F (85% formic acid, Trouw Nutrition, Northwich, Cheshire, UK) was added to the silage at a rate of 3.0 L/tonne. All cows were fed for ad libitum intake (10% refusals) twice a day (0800 and 1600 h) a total mixed diet containing about 60% grass silage and 40% concentrate (DM basis). The four treatments (Table 2) were concentrates based on (i) Megalac (MEG; Volac Ltd., Roston, Hertfor-
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FATTY ACIDS AND REPRODUCTION Table 2. Composition of the total diets (% of DM) based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). Ingredient
MEG
LIN
FIS
OIL
Grass silage Megalac1 Soybean meal, 44% CP Formaldehyde-treated whole linseed Ground barley Molasses Premix2 Fish oil3
61.8 2.8 3.4 0 20.6 10.5 0.9 0
58.8 0 0 6.7 22.2 11.3 1.0 0
58.6 0 2.1 3.4 22.4 11.3 1.0 1.2
55.7 0 4.2 0 25.9 13.3 0.9 0
1
Volac Ltd., Roston, Hertfordshire, UK. Premix contained 400,000 IU of vitamin A/kg, 40,000 IU of vitamin D3/kg, 20,000 IU of vitamin E/kg, 20,000 IU of α-tocopherol/kg, 27% Ca, 4.1% P, 2500 mg/kg of Mn, 2500 mg/kg of Zn, 1500 mg/kg of Cu, 760 mg/kg of Fe, 99 mg/kg of I, 40 mg/kg of Co, and 18 mg/kg of Se. 3 South American herring (I. Spencer and Co., Ltd., Fleetwood, Lancashire, UK). 2
dshire, UK), (ii) formaldehyde-treated whole linseed (LIN), (iii) a mixture (50:50, oil basis) of fish oil and formaldehyde-treated whole linseed (FIS), or (iv) no fat in the concentrate but with 500 g of linseed oil being infused daily into the duodenum at a constant rate over 23 h (OIL). All concentrates (Table 3) were in pelleted form and concentrate plus oil consumption were calculated to result in similar CP, ME, and oil intakes as a percentage of total DMI. A commercial salt of fatty acids (MEG) was used in the control diet (MEG) to make all diets iso-energetic and iso-oil. Whole linseed was treated with formaldehyde (LIN) in an attempt to reduce ruminal biohydrogenation of polyunsaturated fatty acids (PUFA). Concentration of formaldehyde in linseed reached 4% on a protein basis, which meets the
minimum recommendation of 2% made by Scott et al. (1971) to provide maximum protection against microbial hydrogenation and successfully modify milk fatty acid composition. Treatment of linseed was carried out by adding 300 g of formalin per kg of whole linseeds to create pH reversible methylene bridges within the seed. Following treatment, the linseed was held for 5 d in curing vessels to allow methyl linkages to form. The protected linseed was bagged and stored at ambient temperature after the curing phase. A duodenal infusion of linseed oil was used for one treatment (OIL) in order to avoid completely the biohydrogenation of omega-3 fatty acids in the rumen. Fish oil was included in one of the treatments (FIS) because, unlike EPA, negligible amounts of DHA were made from α-linolenic
Table 3. Chemical composition of the four concentrates based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil into the duodenum (OIL).1 Item
MEG
LIN
FIS
OIL
SE
DM, % OM, % of DM ADF, % of DM NDF, % of DM Starch, % of DM CP, % of DM Ether extract, % of DM Energy, Mcal/kg of DM Fatty acids, % of total C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:5 C22:6
87.1 93.1 8.2 21.9 36.2 14.7 6.1 4.33
87.0 94.9 8.3 26.1 35.8 14.1 5.2 4.37
86.9 94.7 8.1 23.3 35.9 14.7 5.4 4.44
87.5 94.4 7.6 24.2 39.4 15.2 4.9 4.15
0.3 0.2 0.1 0.4 1.6 0.2 0.4 0.07
0.92 1.24 43.6 0.18 3.90 29.5 19.0 1.6 0 0
0.32 0.35 14.5 0.16 2.79 17.1 25.4 39.4 0 0
0.71 4.55 24.0 3.85 4.40 20.2 21.9 17.5 2.03 0.86
0.32 0.86 20.6 0.64 1.73 14.1 51.0 10.8 0 0
0.08 0.41 2.8 0.38 0.26 1.5 3.3 3.6 0.21 0.09
1
Mean of 5-wk composite samples that were prepared from three weekly samples. Journal of Dairy Science Vol. 85, No. 4, 2002
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acid by elongation/desaturation pathways (Choi et al., 2000). The mixed treatment attempted to utilize the ability to synthesize EPA and used fish oil to ensure that the milk contained some DHA. Experimental Procedures The experiment was designed as a 4 × 4 Latin square with 4 diets, 4 cows and 4 periods; each period lasted 35 d. Adaptation to diets was from d 1 to 14, fecal and urine collection from d 15 to 21, and transrectal ultrasonography from d 22 to 35. Feed intake and milk yield were measured daily. Milk composition was measured in daily bulk samples prepared for each cow from samples taken on four separate days over the last 2 wk of each period. Milk was collected three times a week to determine progesterone concentrations and confirm the beginning of a new estrus cycle. Feces and urine were collected utilizing the externally applied urine separators and techniques described in detail by Aston et al. (1998). Estrous cycles were synchronized in each period. On d 14, a GnRH agonist (Buserelin, Hoechst-Roussel Vet Ltd., Milton Keynes, UK) was administered (8 µg) i.m. to each cow followed 7 d later by a 25 mg i.m. injection of PGF2α (Lutalyse, Pharmacia and Upjohn Ltd., Corby, Northamptonshire, UK). Regression of the CL was confirmed using ultrasonography. Ovaries of each cow were examined by ultrasonography using a Concept MCV ultrasound scanner equipped with a linear array 7.5 MHz probe (Dynamic Imaging Ltd., Livingstone, Scotland, UK) in mid-morning on each day from d 22 to 35 of each experimental period, which corresponded to d 1 to 14 of the estrus cycle. Size and number of ovarian follicles > 3 mm were recorded on detailed follicular maps. Follicles were grouped into three diameter classes for analyses: class 1 (3.0 to 4.9 mm), class 2 (5.0 to 9.9 mm), and class 3 (≥10 mm). Size and position of CL also were recorded. Duodenal samples were collected over two separate 24-h periods on d 21 and 22. The digesta samples were collected continuously over each 24-h period using automatic digesta samplers (Evans et al., 1981) removing approximately 5% of the digesta passing the cannula. The digesta obtained over each 24-h period for each cow was treated as a separate sample. On d 25, rumen fluid samples were taken before the morning feed and at 0.5, 1, 2, 3, 4, 5, 6, and 9.5 h after the feed was offered. Rumen pH was measured immediately after each sample was collected, and then 50-ml samples were acidified with 2-ml concentrated sulphuric acid and stored at −20°C. On d 34, each cow was fitted with a mammary indwelling catheter. On d 35, blood was withdrawn at 0900 h from the mammary vein into vacutainer (Becton Journal of Dairy Science Vol. 85, No. 4, 2002
Dickinson Vacutainer Systems Europe, Meylan Cedex, France) tubes containing either lithium heparin for determination of glucose, EDTA for NEFA analysis, or no preservative for cholesterol analysis. On d 35 (end of the luteal phase and beginning of follicular phase), an oxytocin challenge (Oxytocin-S; Intervet, Cambridge, UK) was administered (100 IU) to stimulate uterine production of PGF2α as previously described by Oldick et al. (1997). Blood was collected into syringes containing one drop of heparin (100µ/ml in 0.9% saline) at 10-min intervals for 1 h prior to the oxytocin injection, at 10-min intervals for 2 h after the oxytocin injection, and at 20 min-intervals for another 2 h. The plasma was separated and frozen at −20°C for subsequent analysis of 13, 14 dihydro-15-keto-prostaglandin F2α (PGFM), the principle metabolite of PGF2α. Chemical Analysis Feed ingredients were sampled three times each week (daily during the measurement of digestibility) and pooled weekly. All samples were frozen at −20°C for subsequent chemical analyses. Methods of analysis for feed and rumen samples used in our laboratory have recently been described in this journal (Dewhurst et al., 2000). Protein, fat, and lactose in milk were determined by infrared analyzer (Milkoscan 605, Foss Electric, Hillerød, Denmark). Concentrations of progesterone in milk were measured by enzyme immunoassay (Ridgeway Science Ltd., Alvington, UK) in a single batch every week, and intra- and inter-assay coefficients of variation were 4.2 and 7.5%, respectively. Concentrations of plasma glucose (kit No. 6, Sigma-Aldrich Ltd., Poole, Dorset, UK), plasma nonesterified FA (kit 9075401; Wako Pure Chemical Industries, Osaka, Japan), and serum total and HDL cholesterol (kit no. 401 and 352, respectively, Sigma-Aldrich Ltd., Poole, Dorset, UK) were analyzed by colorimetric methods. Fatty acid analysis of milk and feed ingredients was carried out using the preparation method of Sukhija and Palmquist (1988), and FA determination in digesta samples was done according to Scollan et al. (2001). Plasma samples were assayed for PGFM after extraction with acidified diethyl ether by radioimmunoassay (Kaker et al., 1984). Parallelism of a pool from cows was demonstrated for all assays and average recovery, which was calculated by addition of various doses of unlabelled hormones to a pooled sample, varied between 94 and 104% for all assays. The sensitivity of the assay was 25 pg/ml, and the intra- and inter-assay coefficients of variation were 12.6 and 14.3%, respectively. Ammonia and VFA in rumen fluid were determined according to the procedures described by Dewhurst et al. (2000).
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Statistical Analysis
Diet Digestibility
All results were subjected to analysis of variance for a 4 × 4 complete Latin square allowing measurement of residual effects (Cochran and Cox, 1957) with the GLM procedures of SAS (1995). Main sources of variation in the model were cow, period, treatment, and residual effects. Significance was declared at P < 0.05 and a trend at P < 0.10, unless otherwise stated. Data on follicular development and size of CL were analyzed from d 1 to 14 of the estrus cycle as repeated measurements across time. Scheffe’s test was used to determine the effect of treatments on size of the CL, the difference in size between the largest and the second largest follicle, and class and number of follicles after a significant F-test. Data on PGFM were analyzed as repeated measurements across time and also as mean concentrations over the 5-h sampling period, peak value, and area under the curve.
There were small but significant differences among diets in the apparent digestibility of various feed fractions (Table 4). The duodenal infusion of linseed oil had no effect on DM digestibility, in agreement with previous studies involving abomasal infusions of fat (Christensen et al., 1994; Drackley et al., 1992; Oldick et al., 1997). The most notable feature of the digestion results is the significantly lower digestibility of CP and ether extract with the LIN diet, which may reflect the inherent attributes of linseed as well as the effects of the formaldehyde treatment. It seems unlikely that this effect relates to impaired rumen function, since there were no effects on DMI and ruminal concentrations of ammonia N and total and individual VFA (data not shown). Doreau and Chilliard (1997) previously reported that NDF digestibility was greater when cows were given fish oil as observed in the present experiment. The lower digestibility of ether extract in the LIN concentrate may reflect the greater accessibility to rumen microbes and enzymes of fats added in the form of Ca salts, dietary oils, or infusions, compared with lipids that are intimately associated with protein in oilseeds. Biohydrogenation values for linolenic acid were calculated from the first period of a 4 × 4 Latin square according to Wu et al. (1991) and averaged 88.8 and 90.2% for steers fed LIN and FIS, respectively (Choi et al., unpublished data), which would indicate that the formaldehyde treatment was not very effective.
RESULTS AND DISCUSSION Feed Composition The chemical composition of the four concentrates (Table 3) was very similar, with no difference in DM, OM, ADF, starch, CP, or ether extract concentrations. The higher NDF concentration of the LIN diet was expected since NDF is higher in linseed than in the other feed ingredients. The fatty acid composition of concentrates differed among concentrates (Table 3), reflecting our formulation objectives. The concentrations of C16:0 and C18:1 were high for MEG, and C18:3 concentrations were higher for LIN and OIL. Fish oil (in diet FIS) was the only source of EPA and DHA. Feed Intake Total DMI was similar among treatments MEG, LIN, and FIS (Table 4), in agreement with most literature reports that showed little effect of the effects of concentration and type of fat supplement when total fat concentration was below 6% of the DM (Kennelly, 1996; Oldick et al., 1997; Dhiman et al., 2000; Petit et al., 2001). Our results are equivocal on the effect of duodenal infusion of linseed oil on total DMI (which included the amount of oil infused into the duodenum), with no significant effect, but the lowest DMI for cows that received the linseed oil infusion. Other authors showed little effect on DMI of infusing 1.5 kg/d of rapeseed oil into the duodenum of cows in late lactation (Chilliard et al., 1991), though post-ruminal infusion of free fatty acids depressed DMI (Benson et al., 2001; Bremner et al., 1998; Christensen et al., 1994; Drackley et al., 1992).
Milk Production and Composition Milk yield and 4% FCM yield (Table 4) were similar for all treatments, in agreement with literature studies showing that the infusion of mainly unsaturated fats had little effect on milk production (Chilliard et al., 1991; Christensen et al., 1994; Drackley et al., 1992). Dhiman et al. (2000) reported similar milk yield for cows fed either 3.6% soybean oil, 2.2% linseed oil, 4.4% linseed oil, 18% raw cracked soybeans or 18% roasted cracked soybeans, and Cant et al. (1997) demonstrated that feeding 2% fish oil had no effect on milk yield. Moreover, Donovan et al. (2000) reported that cows fed diets of 1% fish oil produced more milk. In addition, Keady et al. (2000) reported that as intake of fish oil increased from 0 to 150 to 300 to 450 g/d, milk production increased. Concentrations of fat and protein in milk were significantly reduced by the inclusion of fish oil in diets (treatment FIS); there was remarkable similarity of milk fat and protein concentrations for treatments MEG, LIN, and OIL. There was a marked reduction in the yield of milk fat from cows that received fish oil. The use of fish oil to depress milk fat percentage is now Journal of Dairy Science Vol. 85, No. 4, 2002
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PETIT ET AL. Table 4. Feed intake, digestibility, covariant-adjusted milk production, and milk composition of Holstein cows fed a total mixed diet containing a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS) or infused with linseed oil into the duodenum (OIL).1 Item
MEG
LIN
FIS
OIL
SE
Total DMI, kg/d Digestibility, % DM CP ADF NDF Ether extract Energy Milk yield, kg/d 4% FCM, kg/d Milk composition, % Fat Protein Lactose Milk components, kg/d Fat Protein Lactose
17.8
17.1
17.2
16.3
0.6
77.4b 70.3a 76.3b 76.0bc 76.0b 77.6bc 22.1 22.5
76.7b 66.8b 75.4b 75.9c 70.3c 76.7c 21.7 21.4
79.7a 72.0a 77.9a 78.8a 79.4ab 79.8a 22.2 18.9
79.0ab 71.5a 76.8ab 77.3b 82.8a 79.1ab 20.9 20.6
0.5 0.7 0.5 0.5 0.6 0.5 1.3 1.2
4.12a 3.25a 4.59b
3.97a 3.28a 4.63b
3.11b 3.08b 4.61b
4.01a 3.30a 4.75a
0.70 0.05 0.04
0.91a 0.72 1.02
0.85a 0.71 1.00
0.67b 0.68 1.02
0.82ab 0.67 0.98
0.05 0.04 0.06
Means within a row with no common superscript differ (P < 0.05). Least squares means with pooled SE.
a,b,c 1
a well-established procedure (e.g., Cant et al., 1997) and likely results from the generation of high levels of trans-FA in the rumen (Baumgard et al., 2000). Higher milk fat percentages for the other treatments likely reflect the lower levels of trans-FA as a consequence of rumen protection as calcium salts (MEG), as seeds (formaldehyde-treatment; LIN) and by post-ruminal infusion (OIL). The addition of polyunsaturated oils in a free form tends to depress milk fat percentage (Selner and Schultz, 1980), whereas supplementation of oil through seeds maintains or increases milk fat content (Mohamed et al., 1988). Although supplementary fat tends to depress milk protein percentage, this has not always been observed (Drackley et al., 1992). The depression of milk protein percentage when cows were given fish oil is in agreement with the results of Cant et al. (1997). It seems that the mechanism involving reduced mammary blood flow that was proposed by Cant et al. (1997) was operating since we found no evidence of a negative impact of the fish oil on feed intake, rumen fermentation, and digestibilities.
these diets, as well as the relatively high levels of C18:3 in the immature, unwilted grass silage used for this study (Dewhurst and King, 1998). Intermediate levels of C18:3 (1.4%) were obtained for the FIS diet, which supplied both linseed oil and fish oil and resulted in a significant increase in the proportion of EPA in milk FA, in agreement with Jones et al. (2000). The most remarkable aspect of these results is the very high level of C18:3 (13.9% of total FA) in milk from cows receiving the linseed oil infusion, demonstrating the large potential to increase omega-3 FA in milk if rumen biohydrogenation can be reduced. The relatively small increase in output of C18:3 for treatment LIN suggests that the formaldehyde treatment was not very effective in reducing rumen biohydrogenation of C18:3, in agreement with results from the analysis of duodenal digesta collected on steers fed similar diets (Choi et al., unpublished data). Increasing the supply of alpha-linolenic acid (diets LIN and OIL) did not result in an increased concentration of EPA in milk. This suggests lower levels of chain elongation and desaturation that were observed when steers were given similar diets (Choi et al., 2000).
Milk Fatty Acids Results of milk FA analysis are presented in Table 5. The proportion of milk FA as α-linolenic acid (C18:3) was relatively high even for the MEG diet in which the concentrates supplied only low levels of C18:3. This likely reflects the relatively high forage proportion in Journal of Dairy Science Vol. 85, No. 4, 2002
Plasma Analysis Plasma concentrations of NEFA (Table 6) were significantly higher for the cows that received linseed oil infusion, in agreement with earlier studies on duodenal infusion of fat (Drackley et al., 1992; Oldick et al., 1997).
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FATTY ACIDS AND REPRODUCTION Table 5. Milk fatty acid composition of Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS) or infused with linseed oil into the duodenum (OIL).1 Item
MEG
LIN
FIS
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:5 SCFA2 MCFA3 LCFA4 Omega-6/omega-3 ratio
5.7 2.3 1.4 3.1 3.8 11.1bc 1.1 36.9a 1.7b 9.6b 20.3b 1.9c 1.0b 0.14b 16.3 50.7a 33.0c 1.72a
5.3 2.3 1.5 3.4 4.0 11.7b 1.0 31.5b 1.6b 11.9a 21.4b 2.3bc 2.0b 0.17b 16.5 45.8b 37.7b 1.06b
(% of total fatty acids) 4.9 2.1 1.3 3.2 4.4 13.2a 1.4 32.9b 3.3a 4.0d 25.0a 2.6b 1.4b 0.26a 15.9 50.8a 33.3c 1.54a
OIL
SE
4.9 2.3 1.6 4.2 5.2 10.3c 0.7 25.5c 0.9c 8.6c 16.3c 5.4a 13.9a 0.15b 18.2 37.5c 44.3a 0.39c
0.4 0.1 0.1 0.1 0.2 0.3 0.1 1.2 0.2 0.8 0.9 0.4 1.4 0.02 0.4 1.4 1.4 0.14
Means within a row with no common superscript differ (P < 0.05). Least squares means with pooled SE. 2 Short-chain fatty acids (4:0 to 12:0). 3 Medium-chain fatty acids (14:0 to 16:1). 4 Long-chain fatty acids (18:0 to 20:5). a,b,c,d 1
This likely reflects the greater lipolytic activity against fats supplied in this way (Gagliostro and Chilliard, 1991). Plasma NEFA concentrations were equally low for MEG and LIN treatments. Petit et al. (2001) previously reported that cows fed MEG had an increase in NEFA plasma concentration over time, whereas those fed LIN maintained similar concentrations; in the present experiment, the Latin square design might not have allowed differences to become apparent as cows were switched to a different diet every 5 wk. Serum total and HDL cholesterol concentrations varied in the same way as plasma NEFA. There was no difference among treatments for serum LDL cholesterol concentration. Increased plasma cholesterol concentrations were previously reported for cows that were infused postrumi-
nally with fat (Drackley et al., 1992), although Christensen et al. (1994) reported no difference. Plasma glucose concentration was lower for cows fed MEG compared to those fed the other diets. An increase in plasma glucose concentration (Table 6) without a concomitant increase in milk production (Table 4) for cows fed LIN, FIS, and OIL suggests a limited capacity for glucose uptake by the mammary gland. Differences in plasma glucose concentrations cannot be explained by differences in VFA patterns, which were similar among treatments (data not shown). Prostaglandin Synthesis The concentration of PGFM, expressed as the average value for all sampling times, tended (P = 0.07) to be
Table 6. Blood composition of Holstein cows fed a concentrate based on Megalac (MEG), formaldehydetreated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil into the duodenum (OIL).1 Item NEFA, µeq/L Total cholesterol, mg/100 ml HDL cholesterol, mg/100 ml LDL cholesterol, mg/100 ml Glucose, mM
MEG
LIN
bc
c
178 361b 203b 158 3.38b
159 329b 205b 124 3.60a
FIS b
190 339b 199b 140 3.58a
OIL a
229 494a 295a 199 3.58a
SE 78 28 18 13 0.06
Means within a row with no common superscript differ (P < 0.05). Least squares means with pooled SE.
a,b,c 1
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PETIT ET AL. Table 7. Reproduction data of Holstein cows fed a concentrate based on Megalac (MEG), formaldehydetreated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL).1 Item
MEG
LIN
FIS
OIL
SE
PGFM, pg/ml Mean Area under the curve Peak
60.7ab 18,240cd 98.4
53.8b 16,187d 77.6
71.0a 20,808c 93.5
58.5b 17,181d 80.2
4.7 1433 14.0
Follicles diameter, mm Largest, F1 Second largest, F2 F1–F2 Class of follicles, no. Small, 3.0 to 4.9 mm Medium, 5.0 to 8.9 mm Large, ≥10.0 mm Total CL diameter, mm
11.6 6.3 5.3
12.7 6.0 6.7
14.4 9.2 4.8
18.2 9.8 8.4
2.9 2.0 1.8
1.6 1.0 0.9 3.7 11.5f
1.6 0.9 0.8 3.3
1.0 1.7 1.2 3.9
1.0 1.0 1.4 3.3
0.3 0.5 0.3 0.5
16.5e
16.1e
11.1f
1.3
Means within a row with no common superscript differ (P < 0.07). Means within a row with no common superscript differ (P < 0.09). e,f Means within a row with no common superscript differ (P < 0.05). 1 Least squares means with pooled SE. a,b c,d
greater for cows that were fed formaldehyde-treated linseed with fish oil than for those receiving either formaldehyde-treated linseed alone or the infusion of linseed oil (Table 7 and Figure 1). Similar tendencies (P = 0.09 and P = 0.11) were observed when PGFM concentration was expressed as the area under the curve or peak concentration in response to the oxytocin challenge. These observations support our hypothesis that in-
creasing the ratio of omega-3 to omega-6 FA will decrease the synthesis of the dienoic PG (PGF2α), though not in the case of the very long-chain omega-3 FA (EPA and DHA). Reduced levels of PGF2α could contribute to improved fertility of cows fed increased levels of alphalinolenic acid (Petit et al., 2001), through reduced luteolysis (Thatcher et al., 1995). The trend to observe greater concentrations of PGFM for cows on diet FIS was unexpected and in contrast to the results of Mattos et al. (2000) who showed reduced plasma PGFM responses for cows fed fish meal. These latter results are not directly comparable, since their control had no supplementary fat. The discrepancy may simply result from the fact that treatment FIS had only a small effect on the omega-6/omega-3 ratio of PG precursors, as exemplified by the small effect on this ratio in milk (Table 5). In fact, other authors (Wu et al., 1996) have found differences between linolenic acid and EPA plus DHA on eicosanoic synthesis, suggesting that marine- and plant-derived omega-3 FA would have a different effect on PG secretion, as observed in the present experiment. Ovarian Function
Figure 1. Mean plasma 13,14-dihydro-15-keto-PGF2α (PGFM) concentrations following an oxytocin challenge on d 15 of a synchronized estrous cycle for lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 4.7. Journal of Dairy Science Vol. 85, No. 4, 2002
There were no significant effects of treatments on the mean numbers or the mean size of follicles (Table 7). However, although the number of class-1 follicles over time was similar among diets (Figure 2), cows on the FIS treatment tended (P = 0.06) to have a greater number of class-2 follicles (Figure 3); the number of class3 follicles was similar among treatments (Figure 4).
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Figure 2. Mean number of class-1 (3.0 to 4.9 mm) follicles during a synchronized estrous cycle of lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 0.3.
Figure 4. Mean number of class-3 (≥10.0 mm) follicles during a synchronized estrous cycle of lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 0.3.
The size of the largest follicle tended (P = 0.08) to be greater for cows on the FIS treatment (Figure 5). It is suggested that follicle dynamics will be influenced by other effects of dietary FA, such as effects on blood cholesterol (Wehrman et al., 1991). As cholesterol is the precursor of all steroids, increased substrate availability may increase follicular steroid synthesis (Carroll et
al., 1990). Moreover, according to Staples et al. (2000), greater induction of mRNAs for PG endoperoxide synthase-2 from uterine biopsy for cows fed linoleic acid would suggest that PUFA alters ovarian and uterine dynamics, although there is no difference in PGFM curves after an oxytocin challenge. In general, fat supplementation changes the numbers of follicle size classes during the early postpartum period in lactating
Figure 3. Mean number of class-2 (5.0 to 9.9 mm) follicles during a synchronized estrous cycle of lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 0.5.
Figure 5. Size of the largest follicle of lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 2.9. Journal of Dairy Science Vol. 85, No. 4, 2002
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PETIT ET AL.
ance was not responsible for effects. Moreover, cholesterol concentrations were similar for all cows, apart from those that received infusions of linseed oil, suggesting that it was not significant in the normal diets of the current and previous experiment. We have shown that the release of PGFM in response to a standard oxytocin challenge was lower for cows in which diets led to a marked decrease in the omega-6/ omega-3 FA ratio in milk (LIN and OIL but not FIS). This could contribute to improved gestation rates, as would the increased CL diameter of cows that received moderate amounts of dietary omega-3 fatty acids (LIN and FIS). ACKNOWLEDGMENTS Figure 6. Size of the corpora lutea (CL) of lactating Holstein cows fed a concentrate based on Megalac (MEG), formaldehyde-treated whole linseed (LIN), a 50:50 (oil basis) mixture of formaldehydetreated whole linseed and fish oil (FIS), or infused with linseed oil in the duodenum (OIL). SEM = 1.3.
dairy and beef cows (Staples et al., 1998). Oldick et al. (1997) used a 4 × 4 Latin square design with procedures similar to those of the present experiment, and they reported that the first dominant follicle of cows infused with yellow grease (17.4% linoleic acid) was larger in diameter than that of cows infused with tallow (2% linoleic acid), as measured using ultrasonography. The mean size of the CL (Table 7) was greatest for cows that received great dietary amounts of omega-3 FA (treatments LIN and FIS), although size of the CL over time did not differ among treatments (Figure 6); greater average CL size might have contributed to the increased conception rate of cows offered LIN as compared to those fed MEG in our recent study (Petit et al., 2001), though we did not find any difference in CL size in this previous experiment. However, it is unclear why cows infused with linseed oil had lower CL size than those fed LIN. CONCLUSIONS We sought to explain positive effects of increasing the supply of omega-3 FA to dairy cows on gestation rates in an earlier experiment (Petit et al., 2001). Positive effects of fats on reproductive function are thought to occur through increased energy balance (Butler and Smith, 1989), increased concentrations of cholesterol and progesterone in plasma (Carroll et al., 1990), changes in follicle dynamics in the ovary (Lucy et al., 1991), and alterations in PG secretion (Wathes et al., 1998). In the present experiment, cows had similar DMI, milk yield, and BW, suggesting that energy balJournal of Dairy Science Vol. 85, No. 4, 2002
The authors thank J. K. S. Tweed, P. J. King, R. T. Evans, V. J. Theobald, and W. J. Fisher for technical assistance and the dairy barn staff at the Trawsgoed farm in Wales for helping maintain the cows. REFERENCES Abayasekara, D. R. E., and D. C. Wathes. 1999. Effects of altering dietary fatty acid composition on prostaglandin synthesis and fertility. Prostaglandins, Leukotrienes and Essential Fatty Acids 61:275–287. Aston, K., W. J. Fisher, A. B. McAllan, M. S. Dhanoa, and R. J. Dewhurst. 1998. Supplementation of grass silage-based diets with small quantities of concentrates: Strategies for allocating concentrate crude protein. Anim. Sci. 67:17–26. Barnouin, J., and M. Chassagne. 1991. An aetiological hypothesis for the nutrition-induced association between retained placenta and milk fever in the dairy cows. Ann. Rech. Vet. 22:331–343. Baumgard, L. H., B. A. Corl, D. A. Dwyer, A. Saebo, and D. E. Bauman. 2000. Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis. Am. J. Physiol.—Reg. Integr. Comp. Physiol. 278:R179–R184. Benson, J. A., C. K. Reynolds, D. J. Humphries, S. M. Rutter, and D. E. Beever. 2001. Effects of abomasal infusion of long-chain fatty acids on intake, feeding behavior and milk production in dairy cows. J. Dairy Sci. 84:1182–1191. Bremner, D. R., L. D. Ruppert, J. H. Clark, and J. K. Drackley. 1998. Effects of chain length and unsaturation of fatty acid mixtures infused into the abomasum of lactating dairy cows. J. Dairy. Sci. 81:176–188. Burr, M. L. 1989. Fish and the cardiovascular system. Prog. Food Nutr. Sci. 13:291–316. Butler, W. R., and R. D. Smith. 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767–783. Cant, J. P., A. H. Fredeen, T. MacIntyre, J. Gunn, and N. Crowe. 1997. Effect of fish oil and monensin on milk composition in dairy cows. Can. J. Anim. Sci. 77:125–131. Carroll, D. J., M. J. Jerred, R. R. Grummer, D. K. Combs, R. A. Pierson, and E. R. Hauser. 1990. Effects of fat supplementation and immature alfalfa to concentrate ratio on plasma progesterone, energy balance, and reproductive traits of dairy cattle. J. Dairy Sci. 73:2855–2863. Chilliard, Y., D. Bauchart, G. Gagliostro, A. Ollier, and M. Vermorel. 1991. Duodenal rapeseed oil infusion in early and midlactation cows. 1. Intestinal apparent digestibility of fatty acids and lipids. J. Dairy Sci. 74:490–498.
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