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,2
* Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, Beeville 78102;
and
Department of Animal Science, Center for Animal Biotechnology and Genomics, Texas A&M University, College Station 77843; and
and
Department of Animal Science, University of Missouri, Columbia 65211
2 Correspondence:
3507 Hwy 59 E (fax: 361-358-4930; E-mail:
glw{at}fnbnet.net).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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Key Words: Cattle • Conjugated Linoleic Acid • Fat • Leptin • Puberty
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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One approach for studying the roles of adiposity and leptin in pubertal development would be to reduce the ratio of fat to lean tissue during early growth and development, thereby reducing leptin availability. In monogastric species, reductions in adipocyte proliferation and circulating leptin may be accomplished by feeding conjugated linoleic acids (CLA), a generic term for conjugated isomers of linoleic (18:2) and linolenic (18:3) acids (Parodi, 1999; Medina et al., 2000). Because CLA are intermediate products of 18:2 and 18:3 biohydrogenation in the rumen, their production and accretion can be increased by feeding diets high in 18:2 and 18:3 to cattle (Parodi, 1999). Importantly, diets high in linoleic acid also increase serum lipoprotein cholesterol, insulin, and GH, and positively modulate ovarian physiology in mature cows (Williams et al., 1998; Williams and Stanko, 2000). However, these effects appear to be unrelated to adiposity or leptin. Herein, we hypothesized that diets high in linoleic acid would increase CLA production, reduce adiposity and serum leptin, and delay puberty.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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Twenty spring-born, crossbred heifers (1/2 Red or Black Angus x 1/4 Brahman x 1/4 Hereford), born within a 30-d period, were utilized. Before weaning and assignment to the study, all heifers received primary and secondary immunizations against common respiratory viruses, leptospirosis, vibriosis, and pasturella using a combination of attenuated live and killed vaccines. Heifers were weaned at approximately 3 mo of age and stratified by age, BW, and breed of sire (Red vs Black Angus) into four pens (five heifers/pen) each measuring 25.9 x 9.5 m2. Body weight stratification resulted in two pens of heavier (heavy) and two pens of lighter (light) heifers averaging (±SEM) 126 ± 4 and 103 ± 4 kg, respectively. At weaning, heifers were fed a diet consisting of 1.3 kg of a concentrate containing 78% sorghum, 20% cottonseed meal, and 1.4% limestone fortified with vitamins A, D, and E, and had ad libitum access to coastal bermudagrass hay. At 4 mo of age, pens of heavy and light heifers were each assigned randomly to receive either a high fat (HF; n =10) or control (C; n = 10) diet until slaughter. Complete mixed diets were isocaloric and isonitrogenous and formulated initially to promote a gain of 0.9 kg/d utilizing NRC recommendations (1984; Table 1
). The HF diet contained added fat equal to 5% of total DM intake, with whole sunflower seed serving as the primary fat source and source of linoleic acid (55% oil, Pope Testing Laboratories, Inc, Dallas, TX; 70% linoleic acid, National Sunflower Association). Diet composition and DM were adjusted for BW gain every 2 to 3 wk until heifers weighed 250 ± 8 kg, at which time diets were adjusted to reduce rate of gain to 0.45 kg/d. At this time, total added fat in the HF group was increased from 5 to 7% of total DM.
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1 ng/mL over two consecutive samples and twice weekly transrectal ultrasonagraphy to visualize corpora lutea. Body weights were recorded weekly until slaughter at 325 ± 10 d of age, at which time all heifers except one in the HF group were pubertal. At slaughter, longissimus muscle between the ninth and 11th rib was excised and analyzed for the percent DM, percentage of protein, total fat (% ether extract), and fatty acid composition. Subcutaneous fat, from the tailhead region, and kidney, heart, and pelvic (KHP) fat were also collected and analyzed for fatty acid composition. RIA and Enzymatic Assays
Circulating concentrations of leptin and progesterone were analyzed using a specific, highly sensitive ovine leptin RIA validated for bovine serum (Delavaud et al., 2000) and the Coat-a-Count assay kit (DPC, Los Angeles, CA), respectively. Use of these assays has been reported previously from our laboratory (Amstalden et al., 2000; Fajersson et al., 1999). Serum insulin, GH, and IGF-I were determined by RIA as described previously (Ryan et al., 1995), except that radiolabeled [125I] porcine insulin (Peninsula Laboratories, Belmont, CA) was utilized as tracer instead of bovine insulin. The bovine insulin antiserum (ICN, Lisle, IL) has a cross reactivity with other related peptides of <0.2% and 100% with human and porcine insulin at 50% displacement. Serum concentrations of total cholesterol were determined by the enzymatic method of Allain et al. (1974) utilizing total cholesterol kit reagents (Sigma-Aldrich, St. Louis, MO) as reported previously (Williams et al., 1989). Intra- and interassay CV for all assays averaged 5 to 9% and 11 to 17%, respectively.
Major Fatty Acid Composition
Adipose and muscle samples were analyzed for fatty acid composition using a method described previously by Folch et al. (1957). Total lipids were extracted from 1 g of muscle and adipose tissue using 2:1 (vol/vol) chloroform:methanol solvent mixture. Total extracted lipids were methylated with 14% boron trifluoride-methanol by the method of Slover and Lanza (1979). Methylated lipids were analyzed using a flame ionization detector on a gas chromatograph using a Varian (Varian, Inc. Walnut Creek, CA) model CP-800, fixed with a CP-8200 (Varian, Inc) autosampler and a flame ionization detector equipped with a 0.25-mm x 100-m fused-silica, silver nitrate-impregnated capillary column (Chrompack, Middelburg, The Netherlands). The injection port and detector temperatures were maintained at 270 and 300°C, respectively. Gas pressures were 2.2 kg/cm2 for the carrier gas (helium), 0.6 kg/cm2 for the make-up gas (nitrogen), and 0.5 kg/cm2 for the combustion air. Chromatograms were recorded with a computing integrator (Shimazu Chromatopac C-R6A). Identification of sample fatty acids were made by comparing the relative retention times of standards previously validated by Zembayashi et al. (1995).
Carcass Composition
Heifers were slaughtered at the Rosenthal Meat Science Center, Texas A&M University, College Station using standard methodology involving captive bolt stunning followed by exanguination. Fat from the tailhead and KHP region were collected at that time. Carcasses were chilled for 48 h after slaughter, at which time longissimus dorsi muscle between the ninth and 11th rib was collected, vacuum-packed, and stored at -10°C until analyses for moisture and total fat content as described previously by Hankins et al. (1946). Percent protein was measured using a Leco N Analyzer (St. Joseph, MI) model FP-2000.
Statistical Analyses
One heifer in the HF light group did not reach puberty within the time allotted for the study (325 ± 10 d of age); therefore, for the purpose of analyzing mean age at puberty, this heifer was assigned the maximal value using the date when the last pubertal heifer was slaughtered. Main effects of diet, BW group, and BW group x diet on age at puberty, percentage of DM, protein, fat, and fatty acid composition were determined with ANOVA using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). Endocrine data were analyzed in two ways using the MIXED procedure of SAS for repeated measures: 1) from the onset of the study to slaughter and 2) for 20 wk normalized to the wk of pubertal ovulation (wk 0). Sources of variation were diet, BW group, week, and interactions among the sources. Heifer within diet and BW classification were used as the "subject" for the MIXED procedure for repeated measures to account for correlated variation within animal. The least squares means procedure was used to compare means when a significant difference was detected in the MIXED analysis. Pearson’s correlation coefficients were determined among the described variables using the CORR procedure of SAS.
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Results |
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Heifers in both the heavy and light BW groups gained 0.88 ± 0.03 kg/d, and weighed 301 ± 7 and 300 ± 11 kg, respectively, at puberty (Table 2). Heifers in the HF heavy group tended (P < 0.10) to reach puberty at an age (307 ± 14 d) older than the HF light, C heavy, and C light groups (280 ± 5, 273 ± 9, and 289 ± 8 d, respectively). One heifer in the HF group did not reach puberty within the time allotted for completion of the study. At slaughter, heifers in the heavy and light BW groups weighed 337 ± 8 and 300 ± 11 kg, respectively. No differences in carcass weight were detected between the HF and C groups (Table 2). Diet did not affect percent DM or fat of longissimus muscle; however, the percentage of total protein tended to be lower in the HF group (Table 2).
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Tissue fatty acid compositions are presented in Table 3. Heifers receiving the HF diet had a higher (P < 0.01) percentage of linoleic acid (18:2) in all tissues analyzed. The cis-9, trans-11 CLA, one of two common and abundant isomers of CLA, was detected in KHP and subcutaneous fat. Content of CLA in KHP was not affected by diet; however, cis-9, trans-11 CLA was higher (P < 0.01) in subcutaneous fat from HF heifers. The second common isomer of CLA, trans-10, cis-12, was only detected in the subcutaneous fat of six heifers (HF = 2; C = 4) and did not differ relative to diet. Diet had no effect on linolenic acid (18:3) tissue content; however, heavy groups of heifers had a higher percentage in KHP fat than light groups (P < 0.05). Heifers receiving HF diets had a higher (P < 0.05) percentage of stearic acid (18:0) in both muscle tissue and KHP fat, but not in subcutaneous fat. Average decreases of 19 and 53%, respectively (P < 0.03), in palmitic (16:0) and palmitoleic (16:1) acids were detected in all tissues analyzed in the HF heifers compared to controls. Myristic acid (14:0) did not differ in longissimus muscle or subcutaneous fat; however, percentage of 14:0 was higher (P < 0.01) in KHP fat of the C heifers.
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Mean serum concentrations of leptin were not affected by diet; however, leptin increased (P < 0.01; Figure 1) linearly in all heifers as puberty approached. Circulating leptin was positively correlated with BW (P < 0.01), total cholesterol (P < 0.01), and insulin (P < 0.05), but negatively associated with serum GH and IGF-I (P < 0.05; Table 4). In contrast to circulating leptin, mean serum concentrations of GH and IGF-I decreased (P < 0.01) beginning approximately 15 wk before puberty (Figure 1). Moreover, circulating IGF-I was lower (P < 0.01) in heifers receiving the HF diet than those on the C diet (136 ± 5 and 183 ± 12 ng/mL, respectively). Although diet did not influence overall mean serum concentrations of GH, a higher (P < 0.02) serum concentration of GH was detected in HF heifers between 15 and 19 wk before puberty compared to controls (Figure 1). Circulating concentrations of insulin and total cholesterol increased throughout the experiment (P < 0.01; Figure 1) in both C and HF heifers. However, total cholesterol was higher (P < 0.01) in the HF group than in the C group (141 ± 3 and 105 ± 4 mg/dL, respectively). Mean serum concentrations of insulin were higher (P < 0.02) in the C light and HF heavy heifers than in the HF light and C heavy heifers, 0.73 ± 0.03 vs 0.63 ± 0.05, respectively. This dichotomy was reflected by a significant (P < 0.02) diet x BW group interaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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Mean serum concentrations of leptin increased as puberty approached. This is consistent with observations reported by Garcia et al. (2002); however, no differences in mean concentrations of leptin were detected between dietary groups. This is likely attributable to similarities in the percentage of total carcass fat in the HF and C heifers. Recent studies in monogastric species have reported that CLA reduces adiposity, which results in a leaner carcass in rodents (West et al., 1998), chickens (Szymczyk et al., 2001), and pigs (Dugan et al., 1997). The fat-reducing effects of CLA are believed to occur by preventing both the proliferation and lipogenic activity of adipocytes (Evans et al., 2000; Loor and Herbein et al., 1998). Such action becomes optimal when exposure to high concentrations of CLA occurs during the early stages of development for an extended period of time (Parodi, 1999).
In the current study, effects of HF diets on lipid metabolism were obvious as shown by increased concentrations of total cholesterol in serum and linoleic acid in muscle and fat samples. Furthermore, the increase in cis-9, trans-11 CLA content in subcutaneous adipose tissue in HF heifers implies that more linoleic acid was available for CLA production. Beaulieu et al. (2002) observed a higher cis-9, trans-11 CLA content in subcutaneous adipose tissue compared to other fat depots in steers; however, CLA content was not affected by soybean oil supplementation, another rich source of linoleic acid. Contrasting effects of high linoleic acid diets on tissue fatty acid composition in cattle may be due to breed type and/or sex, as has been reported by Zembayashi et al. (1995). A further indirect verification of increased CLA production in the HF group of the current work were the observed decreases in tissue content of myristic, palmitic, and palmitoleic acids, which are considered to be hallmarks of increased accumulation of CLA in tissue (Loor and Herbein, 1998). In previous reports, concentrations of CLA increased 109% in milk fat obtained from Holstein cows receiving extruded oilseeds high in linoleic acid (Dhiman et al., 1999). Moreover, steers receiving diets high in linoleic acid had a greater carcass content of CLA than steers fed low quantities of linoleic acid (French et al., 2000). Although CLA accumulation was greater in HF heifers in the current experiment, the concentration clearly was insufficient to promote marked reductions in total carcass fat. Reports of the fat-reducing effects of CLA have arisen mainly from experiments with monogastrics, which were fed large doses of CLA directly, and in ruminants receiving abomasal infusions of CLA. The latter resulted in a 20% reduction in milk fat in Holstein cows (Loor and Herbein, 1998). However, the infusion of an equal amount of linoleic acid decreased milk fat by only 5%. This indicates that the accumulation of CLA from biohydrogenation of linoleic acid in the rumen may not be large enough to create significant effects on carcass adiposity.
Future efforts to manipulate CLA production in ruminants through optimization of the approach described herein may be problematic. Although increasing the amount of dietary linoleic acid to levels greater than that fed in the current study could potentially increase CLA production, consumption of fats or oils in quantities greater than 5% of DM intake can markedly reduce rumen fiber and protein digestibilities (Byers and Schelling, 1988). Moreover, these problems are most pronounced with diets high in polyunsaturated fatty acids (Jenkins, 1993), which are necessary to produce CLA isomers. Rumen digestibility problems can be avoided to some degree by feeding whole oil seeds, such as in the current study, where dietary added fat was increased to 7% of DM. Whole oilseeds may slow the release of oil into the rumen compared to other forms of fat supplementation. Previously, we have fed cattle up to 8% ether extract on a DM basis using whole cottonseed to manipulate metabolic and endocrine variables and no obvious perturbations in rumen function were apparent (Williams, 1989).
Many studies have shown that mature cows fed a diet high in polyunsaturated fat exhibit an array of metabolic, hormonal, and ovarian responses that can enhance reproductive performance (See reviews by Williams, 1998; Williams and Stanko, 1999). Hence the basis for using dietary fat supplementation in cattle has traditionally focused on its positive benefits. Although the goals of the current study were to gain insight into the relative importance of adiposity and leptin in pubertal development through dietary efforts to reduce their presence, some of the same metabolic and hormonal effects of high linoleic acid diets reported in mature cows were expected also in heifers. These include increased total cholesterol, insulin, and GH in serum. Except for total serum cholesterol, HF diets did not affect these variables in the current study, although paradoxically, serum IGF-1 was lower in HF compared to control heifers. Therefore, the growing heifer appears to represent a physiological state in which the typical hormonal changes that are seen in mature cows are not readily observed. Serum insulin increased in all groups over time, which was expected, because circulating concentrations of insulin are positively associated with DM and caloric intake (Bassett et al., 1971). Unlike insulin, serum concentrations of GH and IGF-I, while increasing early in the feeding period, declined throughout much of the 10-wk period preceding puberty in the current study. Circulating concentrations of IGF-I have been reported to be important for sexual maturation in rodents, primates, and heifers (Hiney et al., 1996; Copeland et al., 1982; Armstrong et al., 1992). Immunizing heifers against growth hormone-releasing hormone resulted in a reduction in serum IGF-I and a delay in age at puberty, suggesting that IGF-I may be important to sexual maturation in cattle. However, large variability exists in serum concentrations of IGF-I in prepubertal heifers. Previous studies have reported moderate increases in circulating concentrations of IGF-I during pubertal development (Garcia et al., 2002; Armstrong et al., 1992; Yelich et al., 1996). However, Jones et al. (1991) reported that serum concentrations of IGF-I in prepubertal heifers vary by breed after observing an increase in circulating IGF-I in prepubertal Angus heifers, but a decrease in Charolais and Simmental heifers as puberty approached. It is possible that maximal serum concentrations of GH and IGF-1 are achieved in heifers at different times, depending upon rates of growth and maturation as influenced by both genetics and nutritional environment. As noted previously, overall concentrations of IGF-I were lower in HF compared to C heifers, a result that was not accompanied by a reduction in GH and a relationship not observed previously in mature cows (Williams, 1998). Collectively, our data indicate that feeding diets high in linoleic acid to developing heifers does not result in altered carcass adiposity or a delay in puberty. Moreover, the previously reported metabolic and ovarian effects that are the hallmark of polyunsaturated fat supplementation in mature cows probably do not apply to the developing, prepubertal heifer.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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Footnotes |
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Received for publication July 15, 2002. Accepted for publication September 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsReferences |
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