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* Department of Animal Science, University of Wyoming, Laramie 82071; and
and
Department of Physiology, Colorado State University, Fort Collins 80523
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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metabolite (PGFM) in primiparous beef cows during early lactation. Beginning 1 d postpartum, 18 primiparous, crossbred beef cows (411 ± 24.3 kg of BW) were fed foxtail millet hay at 1.68% of BW (DM basis) and either a low-fat supplement (control: 63.7% cracked corn; 33.4% safflower seed meal; and 2.9% liquid molasses; DM basis) at 0.35% of BW (n = 9) or a supplement (linoleate) containing 95.3% cracked high-linoleate (79% 18:2n-6) safflower seeds and 4.7% liquid molasses (DM basis) at 0.23% of BW (n = 9). Diets were formulated to be isonitrogenous and isocaloric. The linoleate diet contained 5.4% of DMI as fat vs. 1.2% for control. Beginning 1 d postpartum, cattle were bled every 3 d for collection of serum and plasma. Cattle were slaughtered at 37 ± 3 d postpartum for collection of the medial basal hypothalamus, myometrium, endometrium, caruncular tissue, intercaruncular tissue, and oviduct. Feeding linoleate increased (P = 0.001) plasma concentrations of 18:2n-6, 18:2cis-9 trans-11 and total unsaturated fatty acids; however, 18:1trans-11 did not differ (P = 0.19) between treatments. Concentrations of 20:5n-3 in the medial basal hypothalamus tended (P = 0.10) to be greater for cattle fed linoleate. Concentrations of fatty acids in the oviduct were greater (P < 0.05) than in other uterine tissues. Cows fed linoleate had greater (P = 0.05) concentrations of 18:3n-3 in the endometrium and less (P = 0.06) 18:2cis-9 trans-11 in the myometrium than cows fed the control. Supplemental fat increased (dietary treatment x day postpartum, P = 0.01) concentrations of PGFM in serum more in linoleate than control cows from d 3 to 9 postpartum. Lipid supplementation early in the postpartum period altered the fatty acid composition of medial basal hypothalamus, uterine tissue, and serum concentrations of PGFM. The most novel observation was that the oviduct appeared to be the most sensitive tissue to additional dietary linoleic acid, which could potentially influence fertility.
Key Words: beef cattle • brain • fatty acid • linoleic acid • prostaglandin metabolite • uterine tissue
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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and Williams and Stanko (1999) suggested that improved reproduction in cattle consuming supplemental lipids could be related to an increase in the pool of 18:2n-6 available to attenuate PG production. As an early precursor to PG (Funston, 2004), however, a modest increase in metabolizable 18:2n-6 may reduce the life span of the corpus luteum (CL), an event counterproductive to reproductive success (Hess et al., 2005). Increasing intestinal supply of 18:2n-6 by nearly 3-fold (Scholljegerdes et al., 2004) has led to an increase in serum 13,14-dihydro-15-keto PGF2 metabolite (PGFM) in beef cows fed high-linoleate safflower seeds during the postpartum period (Grant et al., 2005). Therefore, it was hypothesized that supplementation with linoleic acid during the postpartum period differentially influences fatty acid composition of tissues crucial to the reestablishment of fertile estrous cycles and concentrations of serum PGF2 metabolite in beef cattle.
We are not aware of reports on fatty acid composition of reproductive tissue in beef cows during postpartum anestrus, but feeding fish products to increase intake of n-3 PUFA has been shown to increase proportions of n-3 PUFA in plasma and caruncular endometrial tissues of nonlactating, estrous cycling beef cows (Burns et al., 2003) and in caruncular tissue of periparturient dairy cows (Mattos et al., 2004).
Our objectives were to evaluate serum PGFM and fatty acid composition of plasma, uterine, and oviductal tissues in primiparous beef cows fed a forage-based diet and supplemental high-linoleate safflower seeds. Fatty acid composition of the medial basal hypothalamus was also evaluated, because this region of the brain may selectively move EFA through the blood-brain barrier (Edmond, 2001) and is one of the regions where pulsatile secretion of GnRH occurs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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All experimental procedures were conducted in accordance with an approved University of Wyoming Animal Care and Use Committee protocol.
Beginning on d 1 postpartum, 18 primiparous, cross-bred (primarily Angus x Gelbvieh) cows (411 ± 24.3 kg of BW and 5.25 ± 0.24 BCS; Wagner et al., 1988) were fed foxtail millet hay daily at 1.68% of BW (DM basis). Cows were assigned randomly to receive either a low-fat supplement (n = 9; 63.7% cracked corn, 33.4% safflower seed meal, and 2.9% liquid molasses, DM basis) fed at 0.35% of BW (control) or a high-linoleate safflower seed supplement [95.3% cracked high-linoleate safflower seeds (79% of total fatty acids as 18:2n-6) and 4.7% liquid molasses; DM basis] fed at 0.23% of BW (linoleate). The diets were formulated to meet nutrient requirements of a 410-kg primiparous beef cow producing 9.4 kg of milk at peak lactation (NRC, 2000) as well as to provide similar daily quantities of N and TDN (Table 1).
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Sampling
Subsamples of supplements and hay were collected twice weekly throughout the experiment. Whole blood was collected via venipuncture of the coccygeal vein every 3 d from d 1 through 33 postpartum. Blood designated for serum was collected into glass Vacutainer tubes (Becton, Dickinson and Co, Franklin Lakes, NJ), whereas plasma was obtained from blood collected into Vacutainer tubes containing Na heparin. Blood samples were immediately refrigerated for 12 h, after which serum or plasma was harvested by centrifugation at 2,000 x g for 30 min.
On d 37 ± 3 d, cattle were transported to a commercial slaughter facility. The medial basal hypothalamus (Moss et al., 1980) and uterine tissues were obtained approximately 15-min postmortem. There was only sufficient sample of medial basal hypothalamus from 3 control cows and 6 linoleate cows, because this tissue was used in additional analyses not presented herein. Upon removal from the carcass, both horns of the uterus were incised. A sample of intercaruncular, caruncular, endometrial, myometrial, and oviductal tissues was collected. An approximate 1 x 1 cm section of intercaruncular and caruncular tissues was sampled (both intercaruncular and caruncular tissues included the endometrium, myometrium, and perimetrium), and as much of the endometrium and myometrium as possible were dissected from the perimetrium. Both oviducts (from the ampula to the uterotubual junction) were trimmed of connective tissue and ommental fat. Samples of the medial basal hypothalamus, oviduct, and uterine tissues were immediately placed into liquid N and subsequently stored at –80°C.
Sample Analysis
Dietary ingredients and feed refusals were analyzed for CP (Leco FP-528, Leco Corp., St. Joseph, MO) and for fatty acids via direct transesterification (Whitney et al., 1999) with methanolic HCl (Kucuk et al., 2001). Plasma, medial basal hypothalami, uterine, and oviductal tissues were lyophilized (Genesis SQ 25 Super ES freeze dryer, Virtis Co., Gardiner, NY) and ground with a mortar and pestle. Approximately 200 mg of freeze-dried plasma and 135 mg of freeze-dried tissue were subjected to direct saponification by incubating in 4.0 mL of ethanol plus 1 mL of 33% KOH in 16 x 125 mm Teflon-lined screw-cap tubes at 85°C for 1 h. Tubes were vortex-mixed every 2 min. Samples were cooled to room temperature, and 1 mL of 12 N HCl and 3 mL of hexane were added to each tube and vortex-mixed. The hexane layers were transferred to clean 16 x 125 mm Teflon-lined screw-cap tubes and dried under a stream of N2 gas.
The resulting tissue fatty acid extracts were then subjected to direct transesterification by incubating the samples with 2.0 mL of HCl in methanol (1.09 M HCl) and 2 mL of methanol including 1 mg of tridecanoic acid methyl ester (Sigma, St. Louis, MO) as internal standard at 85°C for 1 h. Fatty acid methyl esters were extracted in 2.0 mL of hexane and transferred to GLC vials containing a 1-mm bed of anhydrous Na2SO4. Separation of fatty acid methyl esters was achieved by GLC (model 6890 series II, Hewlett-Packard, Avondale, PA) with a 100-m capillary column (SP-2560, Supelco, Bellefonte, PA) and He as a carrier gas at 0.5 mL/min. The oven temperature was maintained at 175°C for 40 min and then ramped to 240°C at 10°C/min. Injector and flame ionization detector temperatures were 250°C. Identification of peaks was accomplished using purified standards (Nu-Chek Prep, Elysian, MN; Matreya, Pleasant Gap, PA). It has been suggested that the weight percentages of CLA may be underestimated when using an acid catalyst to prepare fatty acid methyl esters (Kramer et al., 1997); however, treatment effects have remained intact when acid and alkaline catalysts were compared (Murrieta et al., 2003).
Serum samples were analyzed for concentrations of PGFM by RIA (Silvia and Niswender, 1984), with modifications reported by Grant et al. (2005). The samples were analyzed in a single assay with an intraassay CV of 7.5%.
Statistical Analysis
Data were analyzed by ANOVA as split-plot using the GLM procedure (SAS Inst. Inc., Cary, NC). Dietary treatment effects were tested with the animal within dietary treatment as the error term (error a), whereas the tissue type and dietary treatment x tissue type interactions were tested with residual error (error b). Additionally, the MIXED procedure of SAS was used for analysis of plasma fatty acid concentrations and serum PGFM over the postpartum period. Fixed effects included dietary treatment, days postpartum, and dietary treatment x days postpartum. Animal was used to specify variation among animals using the RANDOM statement, and days postpartum was used as the repeated effect. Animal within dietary treatment was the nested effect using the SUBJECT statement. Using likelihood ratio testing, an AR-1 structure was deemed most appropriate for the within-subjects effects. Pearson correlation coefficients were determined using the CORR procedure of SAS.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Intake of individual and total fatty acids was greater (P = 0.001) for linoleate than control cows (Table 2). Plasma concentrations of 17:0, 18:1cis-9, 20:4n-6, 22:6n-3, and MUFA were not affected (P 
0.19) by dietary treatment (Table 3). A dietary treatment x day postpartum interaction (P < 0.04) was detected for concentrations of 18:0, 18:1trans-11, 18:2n-6, 18:3n-3, SFA, MUFA, PUFA, total unsaturated fatty acids, and total fatty acids in plasma. Except for 18:1trans-11, the nature of interactions did not preclude evaluation of main effects, because the response was attributable to changes in magnitude over time rather than ranking of treatments. For example, plasma concentrations of 18:2n-6 did not differ (P 0.10) between treatments on d 1 to 6, but concentration of 18:2n-6 in plasma was greater (P < 0.05) in linoleate than control cows for most of the postpartum feeding period (Figure 1). The dietary treatment x days postpartum interactions noted for SFA, MUFA, PUFA, total unsaturated fatty acids, and total fatty acids were the result of an interaction noted for 18:0, 18:2n-6, and 18:3n-3, because the values for those variables are a sum of the specific fatty acids in those particular categories.
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noted that intestinal supply of 18:0 and 18:2n-6 was 3.4 and 2.9 times greater in linoleate vs. control treatments. Filley et al. (2000) also reported that provision of supplemental lipids increased proportions of 18:0 and 18:2n-6 in plasma of postpartum beef heifers.
A dietary treatment x day postpartum interaction (P = 0.04) was detected for plasma concentrations of 18:1trans-11. Overall, concentrations of 18:1trans-11 in plasma did not differ (P = 0.19) between treatments even though Scholljegerdes et al. (2004) reported increased duodenal supply of 18:1trans-11 when cattle were fed cracked high-linoleate safflower seeds. The apparent lack of concurrence between duodenal supply of 18:1trans-11 and concentrations of 18:1trans-11 in plasma may indicate that tissues, such as the mammary gland, rapidly remove 18:1trans-11 from circulation (Chilliard et al., 2000; Bauman et al., 2001). Mammary tissue and milk from cows fed linoleate had greater concentrations of 18:1trans-11 than cows fed control (Murrieta et al., 2005). Increased activity of stearoyl-CoA desaturase in the intestinal mucosa (Archibeque et al., 2005) also may preclude detecting differences in plasma concentrations of 18:1trans-11.
Increased (P = 0.001) plasma concentrations of 18:2cis-9 trans-11 in cows fed linoleate suggests that 18:1trans-11 was desaturated by stearoyl-CoA desaturase in the intestinal mucosa. Alternatively, greater concentrations of 18:2cis-9 trans-11 in plasma from cows fed linoleate may reflect increased duodenal supply of this fatty acid (Archibeque et al., 2005). Flow of 18:2cis-9 trans-11 to the duodenum was 0.1 g/d greater in cattle fed linoleate vs. control (Scholljegerdes et al., 2004).
Medial Basal Hypothalamic Fatty Acids
Concentrations of fatty acids in the medial basal hypothalamus did not differ (P 0.17) between treatments with the exception of 20:5n-3, which tended (P = 0.10) to be greater for cows fed linoleate (Table 4). In contrast, MacDonald et al. (1996) noted that rats fed corn oil had greater 18:2n-6 levels in both the plasma and cerebra compared with rats fed beef tallow, but other fatty acids did not differ among dietary treatments. Suarez et al. (1996) also demonstrated that rats consuming a 10% fat diet high in n-3 fatty acids had increased n-3 fatty acids in all tissues including the brain.
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0.17) between treatments for fatty acid concentrations in the medial basal hypothalamus in the present experiment could be due to the relatively small number of samples and the ability of the brain to strictly regulate fatty acid production or fatty acid transport out of the blood (Sperry et al., 1940; Edmond, 2001). The brain can readily synthesize nonessential fatty acids (Sperry et al., 1940) and transport n-6 (Edmond, 2001) and n-3 (Pawlosky et al., 1996) fatty acids via fatty acid transporters that are specific to number of double bonds in the fatty acid. Plasma concentrations of 20:5n-3 increased (P = 0.001) in the linoleate-fed cows, which may have led to an increase in medial basal hypothalamus concentrations of 20:5n-3. Jenkins and Kramer (1990) concluded that dietary 18:3n-3 was the primary source of n-3 PUFA in the brain of calves. The increase in medial basal hypothalamus concentrations of 20:5n-3 for cows fed linoleate also could have resulted from the greater intake of 18:3n-3 and subsequent desaturation and elongation of 18:3n-3 to 20:5n-3, a fatty acid essential to neural development (Youdim et al., 2000).
Interestingly, 18:1trans-11 was a major fatty acid in the medial basal hypothalamus tissue. Although SFA and MUFA are the predominant fatty acids found in brain tissue (Anding and Hwang, 1986; Jenkins and Kramer, 1990), it is not known if isomers resulting from ruminal biohydrogenation, such as 18:1trans-11 or CLA, can pass the blood-brain barrier. Our observations indicate that unique isomers produced through ruminal biohydrogenation can be transported across the blood-brain barrier.
Uterine and Oviductal Fatty Acids
A dietary treatment x tissue type interaction was detected for concentrations of 18:2n-6 (P = 0.01) and 18:3n-3 (P = 0.03; Figure 2). Specifically, supplementation with high-linoleate safflower seeds increased concentrations of 18:2n-6 in the oviduct, but not in other uterine tissues. Furthermore, concentrations of 18:3n-3 in the endometrium were greater, but concentrations of 18:3n-3 in the oviduct were less for linoleate than control cows. Other dietary treatment effects included greater (P = 0.01) concentrations of 15:0 in the myometrium of cows fed linoleate and a trend for greater (P = 0.06) concentrations of 18:2cis-9 trans-11 in the myometrium of cows fed the control. Increased (P = 0.01) concentrations of PUFA in oviducts of cows fed linoleate (16.1 vs. 51.5 mg/g of freeze-dried tissue) were likely due to the increase in concentrations of 18:2n-6 noted previously. Mattos et al. (2004) fed dairy cows either olive oil (56% 18:2n-6) or fish oil (36% 20:5n-3 and 28% 22:6n-3) beginning 21 d before expected parturition and observed greater proportion of 18:2n-6 in cows fed olive oil, whereas feeding fish oil increased 20:5n-3 and 22:6n-3 in caruncular tissue collected 12 h after parturition. Mattos et al. (2004) found that fatty acid composition of dietary oils influenced fatty acid composition of caruncular tissue collected from dairy cows 12 d after parturition. In contrast, Burns et al. (2003) indicated that plasma fatty acid profile did not reflect fatty acid composition of endometrial tissue from nonlactating estrous cycling beef cows. Perhaps the lack of differences between treatments for fatty acid composition in uterine tissues noted in our experiment was due, in part, to the nutrient demands of lactation, which may have reduced the fatty acids available for deposition in uterine tissues. It is also possible that supplements high in 18:2n-6 have less of an effect on fatty acid composition of uterine tissues than fats high in n-3 fatty acids. Elmes et al. (2004) were unable to detect differences in intercaruncular endometrial phospholipid fatty acid composition, but caruncular endometrium phospholipids had greater 20:4n-6 and 22:6n-3 when sheep were supplemented with linoleate in a soybean-based fat product from d 96 to 138 of gestation.
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and PGE2, which may play a major role in oviduct contractility and ovum transport (Spilman and Harper, 1975; Wijayagunawardane et al., 1999, 2001). The proportion of 18:2n-6 within phospholipids of oviducts from Japanese quail has been shown to increase with sexual maturity (Pageaux et al., 1992), and 18:2n-6 has been shown to differentially influence human oviduct contractility in vitro (Lindblom and Andersson, 1985). Implications, however, of the dietary-induced changes in concentrations of arachidonic acid within the oviduct in the current study remain speculative and to be determined.
No differences were noted among tissue types for 15:0 and 20:5n-3 (Table 5). The myometrium had more (P < 0.05) 18:0 and 20:4n-6 than the other uterine tissues and oviduct. The endometrium had the greatest (P < 0.05) concentrations of CLA isomers. Although caruncular and intercaruncular tissue included the endometrium and myometrium, fatty acid concentrations were numerically less than either the endometrium or myometrium alone, probably because of a dilution effect caused by the presence of the tunica serosa. The oviduct had the greatest (P < 0.05) concentrations of 14:0, 16:0, and 18:1trans-11; 18:2n-6; 18:3n-3; and total MUFA, PUFA, and total unsaturated fatty acids of all tissues. Henault and Killian (1993) demonstrated that the oviduct synthesizes and stores a variety of lipids.
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suggested that the majority of dietary fatty acids are utilized by the mammary gland of beef cows through d 60 of lactation. Dietary fatty acids were not used by adipocytes for storage, because neither final BW (406 kg for control vs. 390 kg for linoleate; P = 0.25) nor final BCS (4.9 for control vs. 5.0 for linoleate; P = 0.77) differed between dietary treatments. Additionally, calf ADG was not different (P = 0.15) between dietary treatments (0.66 kg/d for control vs. 0.76 kg/d for linoleate), indicating that milk production did not differ between treatments. The lack of difference in animal ADG and BCS change between treatments was a reflection of the diets being formulated to be isonitrogenous and isocaloric. Nevertheless, cows from both treatments lost 14.5 kg of BW and 0.48 of a BCS. Assuming that 1 kg of BW is equal to 5.82 Mcal (NRC, 2000), energy balance for the experimental period was –2.3 Mcal/d for control and linoleate treatments. Serum PGFM
A dietary treatment x days postpartum interaction (P = 0.01) was observed for PGFM in serum (Figure 3). Serum concentrations of PGFM were greater for the linoleate cows from d 3 to 9 postpartum. Others have also reported similar effects of feeding fats to dairy (Mattos et al., 2004) and beef cows (Lammoglia et al., 1997; Filley et al., 2000). Grant et al. (2005) demonstrated that supplemental high-linoleate safflower seeds increased serum concentrations of PGFM from 25 to 80 d postpartum in multiparous cows.
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reported a negative correlation between caruncular 20:5n-3 and 22:6n-3 and plasma concentrations of PGFM from parturition until d 5 postpartum. In the current study, intercaruncular concentration of 20:5n-3 was negatively correlated (r = –0.83, P = 0.001) with serum concentrations of PGFM on d 33 postpartum. Secretion of PGF2 from bovine aortic endothelial cells in vitro was attenuated by 20:5n-3 (Achard et al., 1997), likely through inhibition of the cyclooxygenase enzyme (Obata et al., 1999), which converts 20:4n-6 to PGF2. Although the endometrium is an important site of PGF2 production, there were no correlations (P = 0.13 to 0.89) between concentrations of PGFM in serum and concentrations of any fatty acids identified in the endometrium. Supplementation with fats high in linoleate results in elevated circulating concentrations of the specific PGF2 metabolite, PGFM, and exerts divergent effects on fatty acid composition of plasma, uteri, oviducts, and the medial basal hypothalamus. Potential reproductive effects of changes in fatty acid composition within tissues remain to be determined.
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Footnotes |
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2 Present address: Northern Great Plains Research Laboratory, USDA-ARS, Mandan, North Dakota.
3 Present address: Department of Animal Science, Purdue University, West Lafayette, Indiana.
4 Corresponding author: brethess{at}uwyo.edu
Received for publication December 16, 2006. Accepted for publication February 18, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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metabolite, and reproductive response in postpartum heifers fed rumen bypass fat. J. Anim. Sci. 78:139–144., and growth hormone in estrous cyclic Brahman cows. J. Anim. Sci. 75:1591–1600., milk composition, and metabolic status of periparturient Holstein cows. J. Dairy Sci. 87:921–932.. J. Anim. Sci. 59:746–753. and endothelin-1 production by cow oviductal epithelial cell monolayers: Effect of progesterone, estradiol 17 ß, oxytocin and luteinizing hormone. Theriogenology 52:791–801.[CrossRef][Medline]This article has been cited by other articles:
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