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* Department of Animal Sciences and
and
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins 80523
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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(i.m.) on d 11 and 25 of supplementation to synchronize estrous cycles. On d 18 postestrus of the second estrous cycle, cows were slaughtered, and caruncular endometrium was dissected from uteri immediately after slaughter. Jugular blood samples were collected immediately before supplementation was initiated (d 0) and at 7-d intervals for 35 d of the study. Plasma eicosapentaenoic and docosahexaenoic acids did not differ between treatment groups on d 0 (P > 0.10); however, these fatty acids were greater in cows supplemented with fish meal over the first 35 d of supplementation compared with cows supplemented with corn gluten meal (P < 0.05). Endometrial docosahexaenoic acid did not differ (P = 0.12), whereas eicosapentaenoic acid was greater (P < 0.05) in cows supplemented with fish meal than in cows supplemented with corn gluten meal. These results indicate that dietary fish meal alters plasma and endometrial n-3 fatty acid composition in beef cows.
Key Words: Cows • Docosahexaenoic Acid • Eicosapentaenoic Acid • Fish Meal • Uterus
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Dunne et al., 2000), costing the U.S. beef and dairy industries millions of dollars annually in lost meat and milk production. Therefore, reducing embryonic mortality is of economic importance.
One cause of embryonic mortality may be that the conceptus (embryo and associated membranes) fails to signal maternal recognition of pregnancy. In the nonpregnant cow, pulses of PGF2 are released from the uterus late in the estrous cycle to cause regression of the corpus luteum (Nancarrow et al., 1973; Kindahl et al., 1976). In the pregnant cow, the conceptus releases interferon-
between 14 and 18 d after conception to reduce uterine PGF2 release, allowing for continued secretion of progesterone by the corpus luteum and establishment of pregnancy. Some conceptuses may not adequately control PGF2 release during this period, resulting in luteolysis and termination of the pregnancy (Thatcher et al., 1994). Therefore, reducing uterine PGF2 synthesis at the time of maternal recognition of pregnancy may improve fertility.
Addition of fish meal to beef cattle diets may alter uterine PGF2 synthesis at the time of maternal recognition of pregnancy. Fatty acids associated with fish meal contain a high percentage of n-3 fatty acids, eicosapentaenoate and docosahexaenoate. Unlike n-6 and n-9 fatty acids in plant and oil seeds, a high percentage of n-3 fatty acids can escape ruminal biohydrogenation and become incorporated in muscle tissue (Ashes et al., 1992). Furthermore, n-3 fatty acids have been shown to inhibit bovine endometrial PGF2 synthesis in vitro and in vivo (Mattos et al., 2001, 2002), allowing for improved fertility. The objective of this study was to determine whether consumption of n-3 fatty acids from fish meal increases plasma and endometrial n-3 fatty acids of beef cows.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Three cows were allotted to separate pens that were being fed a corn silage-based diet supplemented with Menhaden fish meal (Omega Protein, Hammond, LA), and four cows were allotted to separate pens that were being fed a corn silage-based diet supplemented with corn gluten meal. Fish meal and corn gluten meal were supplemented at 5.1 and 8.5% of dietary DM, respectively. Diets were formulated to be isocaloric and isonitrogenous and met or exceeded NRC (1996) requirements for nonlactating beef cows (Table 1). Diets provided 3.2 and 3.5% lipid for corn gluten meal- and fish meal-supplemented cows, respectively; however, the diets differed in fatty acid composition (Table 2). Diets were fed in the morning in amounts that allowed ad libitum intakes throughout the day. Cows were fed their respective diets for approximately 64 d. During the feeding period, average DMI was 11.4 ± 0.5 and 11.8 ± 0.6 kg, respectively, for corn gluten meal- and fish meal-supplemented cows.
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(i.m.; Lutalyse; Pharmacia & Upjohn, Kalamazoo, MI) on d 11 and 25 of the supplemental period to synchronize estrous cycles. Cows completed one estrous cycle (18 to 24 d) before collection of uterine tissue. On d 18 after estrus of the second estrous cycle, cows were transported to the Colorado State University abattoir and slaughtered. Uteri were collected, and caruncular endometrial samples were immediately dissected, snap-frozen in liquid nitrogen, and stored at -70°C until determination of fatty acid composition via GLC. Blood samples were collected via jugular venipuncture immediately before supplementation began (d 0) and at 7-d intervals for the first 35 d of supplementation into tubes containing EDTA. Samples were stored on ice, transported to the laboratory, and centrifuged at 1,500 x g for 20 min at 4°C. Plasma was collected and stored at -70°C until determination of fatty acid composition via GLC.
Fatty Acid Analysis
Long-chain fatty acids in feed samples were methylated using a direct methylation procedure, as described by Sukhija and Palmquist (1988). The same procedure was used to methylate long-chain fatty acids in plasma and endometrial tissue with modification. Briefly, 1 mL of freeze-dried plasma or 1 g of minced caruncular endometrial tissue was placed in 15 mm x 155 mm test tubes. Three milliliters of 5% (vol/vol) methanolic HCl solution was added to each tube. Tubes were then vortexed at low speed for 1 min before incubation in a water bath without shaking. Tubes were incubated at 70°C for 2 h to methylate fatty acids. Following incubation, 7.5 mL of 6% (wt/vol) KCO3 and 1 mL of hexane were added to each tube and vortexed. Tubes were then centrifuged at 2,300 x g for 10 min at room temperature. The organic phase was carefully removed and transferred to a 1.5-mL amber GLC vial (Agilent Technologies, Wilmington, DE), placed in nitrogen evaporator (Organomation Associates, Inc., Berlin, MA), and dried under a stream of N2. Samples were reconstituted in hexane to 0.5 mL and subjected to GLC.
An Agilent 6890 Series gas chromatograph (Agilent Technologies, Wilmington, DE) fixed with a 6B90 series injector and flame ionization detector was used to determine fatty acid composition of feed samples and plasma. The instrument was equipped with a 100-m x 0.25-mm (i.d.) fused silica capillary column (SP-2560; Supelco, Inc., Bellefonte, PA). Fatty acid methyl ester preparations were injected (1 µL) using the split mode. The carrier gas was helium and the split ratio was 100:1 at 180°C. The oven temperature was programmed from an initial temperature of 140°C (0 min) to a final temperature of 225°C at the rate of 2.8°C/min. The final temperature was then held for 18 min. Chromatographs were recorded with a computing integrator (ChemStation Plus chromatograph manager; Agilent Technologies). Standard fatty acid methyl ester mixtures were used to calibrate the gas chromatograph system using reference standards KEL-FIM-FAME-5 (Matreya Inc., State College, PA). Identification of the fatty acids was made by comparing the relative retention times of fatty acid methyl ester peaks from samples with those of standards. Fatty acid data were calculated as normalized area percentages of fatty acids.
Fatty acid composition of endometrial tissue was determined by GLC using a Hewlett-Packard (Avondale, PA) 5890 Series II gas chromatograph fixed with an autoinjector and flame ionization detector. The instrument was equipped with a 30-m x 0.25-mm (i.d.) fused silica capillary column (SP-2380; Supelco Park Inc.). Two microliters of fatty acid methyl ester preparations was injected onto the column using the split mode. The carrier gas was helium and split at 20:1 at 250°C. The oven temperature was maintained at 170°C for 28 min with a flow rate of 1.0 mL/min. The detector temperature was set at 280°C. Individual fatty acids were identified by comparison of retention times with standard methyl fatty acids as described above.
Statistical Analysis
The effect of dietary supplements on plasma fatty acid composition was analyzed using PROC MIXED of SAS (SAS Inst., Inc., Cary, NC) with repeated measures. Heterogeneous autoregressive covariance structure was used in the analysis. The statistical model included treatment, cow, time, and treatment x time as sources of variation. Cow within treatment x time was used as a random variable in the model. If main effects or the interaction were significant (P < 0.05), means were separated using the PDIFF option of SAS. Effect of dietary supplement on caruncular endometrial fatty acid composition was analyzed using the PROC TTEST of SAS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. There was no effect of treatment (P > 0.10), time (P > 0.10), or treatment x time interaction (P > 0.10) for plasma palmitic acid (Figure 1, Panel A). There was no effect of time (P > 0.10) or treatment x time interaction (P > 0.10) for plasma stearic acid (Figure 1, Panel B); however, there was a main effect of treatment (P < 0.05). Plasma stearic acid was greater in cows supplemented with corn gluten meal compared to those cows supplemented with fish meal (P < 0.05).
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. There was no treatment effect on oleic acid (P > 0.10; Figure 2, Panel A); however, there was an effect of time (P < 0.05) and a treatment x time interaction (P < 0.05). Plasma oleic acid decreased (P < 0.05) for both supplement groups over the first 35 d of the supplemental period. The decrease in plasma oleic acid was greater for those cows supplemented with corn gluten meal than for cows supplemented with fish meal (P < 0.05). There was an effect of treatment (P < 0.05), time (P < 0.05), and treatment x time interaction (P < 0.05) for plasma linoleic acid (Figure 2, Panel B). Plasma linoleic acid increased for both supplement groups over the first 35 d of the supplemental period (P < 0.05). This increase in plasma linoleic acid was greater for cows supplemented with corn gluten meal than for cows supplemented with fish meal (P < 0.05). There was no treatment x time interaction for plasma arachidonic acid (P > 0.10; Figure 2, Panel C); however, there was an effect of treatment (P < 0.05) and time (P < 0.05). Plasma arachidonic acid was greater in cows supplemented with fish meal compared to cows supplemented with corn gluten meal (P < 0.05). Plasma arachidonic acid decreased over the first 21 d of the supplemental period followed by an increase from d 28 through 35 (P < 0.05).
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. There was no effect of treatment for plasma linolenic acid (P > 0.10; Figure 3, Panel A). There was an effect of time (P < 0.05) and there tended (P = 0.08) to be a treatment x time interaction. There was a dramatic decrease in plasma linolenic acid over the first 7 d of supplementation for both supplement groups (P < 0.05). Plasma linolenic acid tended (P = 0.08) to be greater for cows supplemented with fish meal during the sampling period. There was an effect of treatment (P < 0.05), time (P < 0.05), and treatment x time interaction (P < 0.05) for plasma eicosapentaenoic acid (Figure 3, Panel B). Plasma eicosapentaenoic acid decreased in cows supplemented with corn gluten meal, whereas plasma eicosapentaenoic acid increased in cows supplemented with fish meal over the first 35 d of the supplemental period (P < 0.05). There was an effect of treatment (P < 0.05), time (P < 0.05), and treatment x time interaction (P < 0.05) for plasma docosahexaenoic acid (Figure 3, Panel C). Plasma docosahexaenoic acid remained unchanged for cows supplemented with corn gluten meal, while plasma docosahexaenoic acid increased in cows supplemented with fish meal over the first 35 d of the supplemental period (P < 0.05).
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. Dietary supplementation had no effect on endometrial palmitic, palmitoleic, oleic, or linolenic fatty acid composition (P > 0.10). Endometrial docosahexaenoic acid did not differ (P = 0.12), whereas eicosapentaenoic acid was greater (P < 0.05) in cows supplemented with fish meal than in endometrium from cows supplemented with corn gluten meal. Furthermore, total n-3 fatty acids were greater (P < 0.05) in endometrial tissue from cows supplemented with fish meal. However, stearic, linoleic, and arachidonic acid composition were less (P < 0.05) in endometrium obtained from cows supplemented with fish meal compared to endometrium obtained from cows supplemented with corn gluten meal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Burke et al., 1997). In addition to being a source of ruminally undergraded protein, fish meal is also a rich source of n-3 fatty acids (i.e., eicosapentaenoic and docosahexaenoic acids). Unlike unprotected n-6 and n-9 fatty acids that can undergo extensive ruminal biohydrogenation, unprotected n-3 fatty acids appear to be more resistant to biohydrogenation by the ruminal microbes (Ashes et al., 1992). In the present study, the addition of fish meal to the diet increased plasma eicosapentaenoic and docosahexaenoic fatty acids, which agrees with our previous studies wherein fish meal supplementation increased plasma n-3 fatty acid composition (Burns et al., 2002a,b). These data support the results of Ashes et al. (1992) in that unprotected n-3 fatty acids escape ruminal biohydrogenation and increase in plasma. However, plasma linolenic acid decreased in both supplement groups in the present study. Cows were fed a corn silage-based diet and silages are generally low in linolenic acid compared to the linolenic acid in grasses (Abayasekara and Wathes, 1999). Furthermore, both dietary supplements provided very little linolenic acid (Table 2
), which may explain the decline in this fatty acid. We have observed similar declines in plasma linolenic acid in cows fed corn silage-based diets low in linolenic acid (Burns et al., 2002a) but not in cows grazing native range high in linolenic acid (Burns et al., 2002b).
There were considerable differences in plasma stearic, oleic, and arachidonic acids between the two dietary supplement groups. As with the changes in plasma n-3 fatty acids, these differences can be best explained by the fatty acid composition and level of intake of the supplements that were being offered to the animals (Table 2). For example, the corn gluten meal supplement contained a greater percentage of linoleic acid compared to the fish meal supplement. Supplementing a greater percentage of linoleic acid to the cow resulted in a higher percentage in the blood (Figure 2; Panel B).
Caruncular endometrium obtained from cows supplemented with fish meal contained a greater percentage of n-3 fatty acids and a lower percentage of arachidonic acid when compared to caruncular endometrium obtained from cows supplemented with corn gluten meal. Rats fed diets high in n-3 fatty acids for 3 wk had greater incorporation of n-3 fatty acids into uterine lipids compared to rats fed diets high in n-6 fatty acids (Howie et al., 1992). Furthermore, arachidonic acid was lower in uterine tissues from rats fed diets high in n-3 fatty acids. The present results show that n-3 fatty acids that are available in plasma may become incorporated into uterine tissues. However, the classes of lipid (phospholipids, triglycerides, etc.) into which the n-3 fatty acids become incorporated remain to be determined.
The addition of fish meal to the diet has been reported to increase fertility in lactating dairy (Bruckental et al., 1989; Armstrong et al., 1990; Burke et al., 1997) and beef (Burns et al., 2002a) cows, possibly by reducing uterine PGF2 synthesis at the time of maternal recognition of pregnancy (Mattos et al., 2001, 2002). However, the mechanism(s) by which fish meal decreases uterine PGF2 synthesis is unclear, although there are a number of ways fish meal supplementation may alter PG metabolism.
Arachidonic acid is the precursor to the 2-series PG, and there is an increase in arachidonic acid in membrane phospholipids before acute PG synthesis in endometrial tissues (Zhang et al., 1995; Meier et al., 1997). In the present study, fish meal supplementation resulted in a greater percentage of n-3 fatty acids incorporated into endometrial tissue presumably at the expense of arachidonic acid. A potential decrease in available arachidonic acid in membrane phospholipids may partially explain the decrease in acute uterine PGF2 synthesis in dairy cows supplemented with fish meal (Mattos et al., 2002). The n-3 fatty acids may also alter the amount of arachidonic acid in endometrial membrane phospholipids by decreasing synthesis of arachidonic acid from linoleic acid. Desaturase and elongase enzymes convert linoleic acid to arachidonic acid. However, both eicosapentaenoic and docosahexaenoic acids have the ability to suppress the synthesis of arachidonic acid by competing more successfully than linoleic acid for these enzymes (Hagve and Christophersen, 1984; Barham et al., 2000).
The n-3 fatty acids may affect cyclooxygenase gene expression or activity resulting in lower PGF2 synthesis in bovine endometrial tissue. Cyclooxygenase is the key regulatory enzyme that converts arachidonic acid to the 2-series PG. Eicosapentaenoic acid can compete with arachidonic acid for the binding sites on the cyclooxygenase enzyme and result in an increase in the 3-series PG (Mattos et al, 2000). In general, the 3-series PG are often less biologically active than the 2-series PG (Needleman et al., 1979). Therefore, an increase in PGF3 may delay regression of the corpus luteum and increase the probability of embryonic survival. The n-3 fatty acids may also alter PG metabolism by affecting cyclooxygenase gene expression. Acute uterine PGF2 synthesis is associated with an increase in cyclooxygenase mRNA (Burns et al., 1997; Fuchs et al., 1999). The n-3 fatty acids have been reported to inhibit cyclooxygenase gene expression in a number of tissues that secrete PG in response to acute stimuli (Gilbert et al., 1999a,b; Obata et al., 1999). Thus, the n-3 fatty acids in fish meal may decrease cyclooxygenase gene expression and reduce PGF2 synthesis in bovine endometrium. However, Mattos et al. (2001) reported that n-3 fatty acids decreased phorbol ester-induced PGF2 synthesis in bovine endometrial cells, but had no effect on the relative abundance of cyclooxygenase mRNA. Further research is required to determine the mechanisms by which n-3 fatty acids affect PGF2 synthesis in bovine endometrial tissues.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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synthesis during maternal recognition of pregnancy, resulting in loss of the pregnancy. Results of the current study show that fish meal supplementation increases endometrial n-3 fatty acids of beef cows. This increase in endometrial n-3 fatty acids may suppress uterine prostaglandin F2 synthesis during the period of maternal recognition of pregnancy and potentially decrease embryonic death loss. Therefore, the addition of fish meal to the diet may be a practical means of improving reproductive efficiency in beef cattle.
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Footnotes |
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2 The authors thank D. Davidson for care and feeding of the animals and D. Abbey and T. Bonnette for collection of data. A special thanks to J. Johnson of Omega Protein (Hammond, LA) for the donation of fish meal and inputs on the design of this experiment.
3 Correspondence: 208B Animal Sciences (phone: 970-491-6649; fax: 970-491-5326; E-mail: pburns{at}ceres.agsci.colostate.edu).
Received for publication May 1, 2003. Accepted for publication July 16, 2003.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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-linolenic acid-supplemented diets prevents serum arachidonic acid accumulation in humans. J. Nutr. 130:1925–1931.
-3 fatty acid profiles in primiparous, lactating beef cows. Prof. Anim. Sci. 18:373–379. during the normal oestrous cycle and early pregnancy in heifers. Acta. Endocrinol. 46:134–149.
from bovine endometrial (BEND) cells stimulated with phorbol 12,13 dibutyrate (PDBU). Theriogenology 55:90.
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