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ANIMAL NUTRITION |
Department of Animal Sciences, The Ohio State University, Wooster
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: beef • immunity • marbling • vitamin A
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Interestingly, the mechanisms controlling the site of fat deposition within the carcass still are not well understood.
Feeding low vitamin A diets to growing cattle appears to increase i.m. without affecting s.c. fat deposition (Gorocica-Buenfil et al., 2007a,b,c). It remains unclear whether extending the duration of vitamin A restriction may further increase i.m. fat deposition. Another important aspect yet to be determined is the effect of vitamin A restriction on cattle health and immune status. Adequate vitamin A status is required to maintain normal immune function (Semba, 1998). In our previous experiments (Gorocica-Buenfil et. al, 2007a,b,c), we have not observed any adverse effects of low vitamin A diets on animal health or performance. However, the ability of vitamin A-restricted steers to respond to an immune challenge has not been evaluated previously.
Modifying the fatty acid composition of beef and increasing its CLA content may benefit human health (McGuire et al., 1999). We hypothesized that feeding low vitamin A diets may increase beef CLA content due to the apparent inhibitory effect of retinol on stearoyl-CoA desaturase (SCD) enzyme activity (Siebert and Zurk, 2004). The effect of a prolonged dietary vitamin A restriction in beef steers has not been evaluated. The objective of the present experiment was to determine the effect of a long restriction of dietary vitamin A in beef steers on the following: 1) animal performance, health, and immunity; 2) carcass characteristics and site of fat deposition; and, 3) fatty acid composition and CLA content of beef.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). A cattle performance experiment was begun in October 2005 at The Ohio State University feedlot in Wooster. Sixty-eight Angus-based steers (224.0 ± 7.6 kg of BW and approximately 7 mo of age) were penned and fed individually in a totally enclosed feedlot barn (slatted concrete floor; metal gates) during the experiment. Pens were 2.6 x 1.5 m (3.9 m2 of floor space per steer).
Six weeks before arrival at the feedlot, steers were vaccinated for infectious bovine rhinotracheitis, parain-fluenza-3, Haemophilus somnus, Pasteurella, and Clostridia (Quadraplex, Somnugen 2P, and Dybelon, respectively, Bioceutic, St. Joseph, MO) and treated for parasites with Ivomec Pour-On (Merial, Duluth, GA). Steers were revaccinated 14 d later. Before initiation of the experiment, all steers received a 65% concentrate diet for a 42-d receiving period.
The experiment had a completely randomized design, and after the receiving period, steers were allotted randomly to 1 of 2 treatments: low vitamin A (LOW, no supplemental vitamin A) or high vitamin A (HIGH, diet supplemented with 2,200 IU/kg of DM; North American Nutrition Companies Inc., Lewisburg, OH). The dietary recommendation for vitamin A is 2,200 IU/kg of feed (NRC, 1996). Considering vitamin A equivalents of dietary ingredients along with supplemental vitamin A, our diets were below and above this recommendation. The diets contained (DM basis) 60% high-moisture corn, 20% roasted soybeans, 10% corn silage, and 10% of a protein, mineral, and vitamin supplement (Table 1). Roasted soybeans were included to provide a source of linoleic acid for the synthesis of CLA.
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). Feed restriction (programmed feeding) was accomplished by feeding a prescribed amount of feed to achieve a gain of 1.1 kg/d (NRC, 1996). Steers were weighed every 2 wk to adjust the amount of feed delivered based on BW for that period. After 141 d of programmed feeding, steers were offered feed for ad libitum intake until the end of the experiment on d 216. Thus, steers were fed ad libitum during the last 75 d of the experiment. At the beginning and the end of the experiment, the steers were weighed on 2 consecutive days to determine the initial and final BW. For all weighing procedures, steers were weighed before feeding at 0800; feed and water were not withheld.
Steers were offered feed once daily beginning at 0800. Daily intake was recorded, and feedstuff samples were taken weekly to adjust dietary DM and to determine DMI. Dietary samples were taken every 2 wk and composited at the end of the experiment for nutrient analyses. Composite feed samples were freeze-dried, ground to pass a 1-mm screen, and analyzed for DM (102°C for 24 h), OM, and N (AOAC, 1996).
Samples of high-moisture corn, corn silage, and the protein supplement were taken every 2 wk for pro-vitamin A carotenoid (β-cryptoxanthin and 
- and β-carotene) analysis. Samples were taken from the feed mixer immediately before ingredients were mixed for the day’s feeding. Samples were put into plastic bags, kept in the dark to avoid carotenoid light damage, and stored frozen at –20°C until carotenoid analyses were performed. Carotenoid analysis was performed as described by Gorocica-Buenfil et al. (2007a).
Jugular blood samples (10 mL) were taken from all steers on d 1, 97, 141, and at slaughter to determine serum retinol. Tubes containing blood samples were wrapped immediately in aluminum foil to avoid retinol light damage and were kept on ice until reaching the laboratory for serum separation. Serum was obtained by centrifuging the blood samples at 2,200 x g at 4°C for 10 min. Samples were frozen at –20°C until vitamin A analysis was performed by HPLC. Hepatic vitamin A stores also were determined. Liver samples were taken at slaughter and were placed immediately on ice and protected from light damage. Samples were stored at –20°C. Hepatic and serum vitamin A levels were analyzed by HPLC, as described by Gorocica-Buenfil et al. (2007c). All trans retinol obtained from Sigma Chemical Co. (St. Louis, MO) was used as the standard.
Effects of dietary vitamin A status on animal health and immunocompetency were determined. Steers were monitored daily for signs of respiratory disease (nasal mucus discharge, coughing, and rapid breathing). Body weight was monitored at 14-d intervals to identify steers that might have lost BW. Any animal exhibiting the above signs and having a body temperature above 39.7°C was treated with antibiotics. Although blindness was not anticipated, steers were monitored daily for signs of blindness (watery, swollen eyes; disoriented). All health problems were recorded.
Antibody response to an ovalbumin vaccine was measured after 140 d on the experimental diets. On d 141, ten steers per treatment were selected randomly, and 10-mL blood samples were taken from the jugular vein. After blood collection, these steers were vaccinated with ovalbumin and were revaccinated 21 d later. Vaccine was prepared by suspending ovalbumin (ovalbumin grade V, Sigma-Aldrich, St. Louis, MO) in PBS, filtering (0.45-µm pore size), and mixing an equal volume with aluminum hydroxide gel adjuvant (Alhydrogel 1.3%, Brenntag Biosector, Frederikssund, Denmark). Final concentration of ovalbumin was 1 mg/mL. All vaccinations were administered s.c. in the neck at a volume of 2 mL. On d 183, ten-milliliter blood samples were taken from the jugular vein of the vaccinated steers. Serum was obtained as described previously. Serum ovalbumin antibody titers were determined by ELISA (Lin et al., 1998). The coating antigen for the ELISA was ovalbumin suspended in 0.01 M ammonium acetate and 0.01 M ammonium carbonate (pH. 8.2) to a final concentration of 200 ng/mL. Procedures for determining end point titers to a protein in bovine serum were detailed by Lin et al. (1998). Bovine immunoglobulin G was determined by using rabbit anti-bovine immunoglobulin G (Sigma Chemical Co.). Titer data were expressed as the reciprocal of the base-2 logarithm of the dilution. Seroconversion was confirmed by comparison of the titer data before and after vaccination.
Steers were slaughtered after 216 d on feed, when the average 12th-rib backfat (BF) thickness was estimated visually to be approximately 1.27 cm. Hot carcass weight, BF thickness, LM area, and KPH were determined by qualified Ohio State University personnel. Carcass yield grade was calculated (USDA, 1997). Quality grade and marbling score were determined by a USDA official. Carcass characteristics (except HCW) were measured after a 48-h chill.
Longissimus muscle samples from the 11th to 12th thoracic rib were collected, trimmed of external fat, ground (model #4822, Hobart Co., Troy, OH), and analyzed for moisture, protein, and ether-extractable lipid (EE) content (AOAC, 1996). Additionally, fatty acids in s.c. adipose tissue were extracted and methylated by alkaline transesterification and analyzed as described by Kramer et al. (1997). Methyl esters of fatty acids were separated on a 0.25 mm x 100 m fused silica column (SP-2560, Supelco Inc., Bellefonte, PA), using a Hewlett-Packard 5890 gas chromatograph with automated injection and data reduction (HP 3365 Chemstation software, Hewlett Packard Co., Santa Clarita, CA). Fatty acid standards were obtained from Nu-Chek Prep Inc. (Elysian, MN). Standards for the CLA isomers cis-9, trans-11; trans-10, cis-12; and trans-9, trans-11 were obtained from Matreya Inc. (Pleasant Gap, PA).
Experimental data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). Serum retinol data were analyzed in a completely randomized design with repeated measures. The model included terms for vitamin A level, days on feed at the time of sample collection, and their interaction. The error structure used was compound symmetry, because it resulted in the lowest Bayesian criteria. Time effects were partitioned into linear, quadratic, and cubic contrasts.
Performance, carcass characteristics, immune response, and fatty acid composition data were analyzed as for a completely randomized design. The model included vitamin A treatment effect. Steer was used as the experimental unit for all statistical analyses. For analysis of quality grade distributions, dummy variables were assigned before statistical analysis (i.e., if a carcass was Select, it would have a value of 1 for Select and a value of 0 for Choice, Prime, etc.).
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. The basal diet provided the equivalent to 1,270 IU of vitamin A/kg of DM, about 60% of the NRC (1996) recommendation. Thus, in total, the HIGH diets provided 3,500 IU of vitamin A/kg of DM. Important differences in the vitamin A content of dietary ingredients between the NRC (1996) feedstuffs data and the actual pro-vitamin A carotenoid content were observed.
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), and G:F was not affected (P > 0.10) by vitamin A restriction during the growing or for the total feeding period. During the finishing phase, a slight tendency for a reduction in G:F was observed in LOW vs. HIGH steers (P = 0.10). However, this difference was less than 5%, and in our previous experiments, feeding low vitamin A diets did not affect G:F (Gorocica-Buenfil et al., 2007a,b,c).
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). Marbling score and quality grade were not affected (P > 0.10) by feeding low vitamin A diets. These results were not expected. In our previous experiments, feeding low vitamin A diets increased marbling and quality grade without affecting s.c. fat and yield grade (Gorocica-Buenfil et al., 2007a,b). In those previous experiments, Angus-based steers were fed for 168 and 145 d, and we speculated that feeding a vitamin A-restricted diet for a longer period of time may further increase the beneficial effects of low vitamin A diets on marbling. Our results do not support this hypothesis. Although the reasons for this discrepancy are unknown, it can be speculated that the inclusion of roasted soybean in the diets may have affected marbling and therefore masked the vitamin A restriction effects. When roasted soybeans were fed to Angus-based steers for 168 d, it appeared that hyperplasia was stimulated in the i.m. depot based on the i.m. adipose cellularity data (Gorocica-Buenfil et al., 2007b). This occurred regardless of the dietary vitamin A concentration. However, in that experiment, roasted soybean inclusion did not affect marbling score, quality grade, or i.m. fat. It can be speculated that the pro-adipocyte differentiation stimulus of feeding roasted soybean for 216 d was such that the i.m. fat depot in both restricted and non-vitamin A-restricted steers was increased. The presumed proliferative stimulus for adipocytes in the i.m. depot by roasted soybean inclusion deserves further research. The time steers remained on feed may have also influenced i.m. fat deposition. Both LOW and HIGH steers in this experiment had greater BF than in our preceding experiments. Differences in marbling between HIGH and LOW steers may have achieved a maximum at some point before slaughter, and then differences may have been lost as i.m. fat deposition of HIGH steers increased with increasing days on feed. Intramuscular fat deposition increases as time on feed increases, but at a decreasing rate (Van Koevering et al., 1995). In a previous experiment, we reported that marbling increased as duration of dietary vitamin A restriction increased (Gorocica-Buenfil et al., 2007c). Furthermore, roasted soybean inclusion in both HIGH and LOW diets may have had a greater effect on marbling than the time steers remained on feed. Marbling score is a variable affected by many factors. Based on our previous data and despite the lack of response in this experiment, we believe that dietary vitamin A concentration is one of those factors.
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). In that experiment, steers were fed for ad libitum intake for the duration of the experiment. The potential interaction between limit feeding and vitamin A restriction to modulate adipocyte differentiation in the i.m. fat depot warrants further research. The effects not only of duration but also of the timing of vitamin A and energy restriction during the feeding period on marbling and adipocyte development are questions that remain to be answered. The CP content of LM was decreased (P < 0.01) slightly by vitamin A restriction (LOW = 19.5 vs. HIGH = 20.6, SEM = 0.23). However, LM area was not affected by dietary vitamin A. Thus, it is unlikely that the small difference in LM protein content has any biological relevance.
Vitamin A restriction decreased (P < 0.01) serum retinol levels over time (vitamin A x days on feed, linear contrast; Figure 1). At the beginning of the experiment, both LOW and HIGH steers had similar levels (LOW = 28.7 vs. HIGH = 30.3 µg/mL, P = 0.20), but by d 97, LOW steers had lower (P < 0.01) retinol levels than HIGH steers. This difference increased as days on feed increased, and by the end of the experiment, LOW steers had 48% lower serum levels than HIGH steers (LOW = 18.7 vs. HIGH = 35.7 µg/mL, P < 0.01). Changes in serum retinol reflected the differences in hepatic retinol stores at slaughter (LOW = 6.3 vs. HIGH = 38.1 µg/g, P < 0.01). These differences suggest that vitamin A stores in the liver were nearly depleted and affected serum retinol (Blaner and Olson, 1994). Feeding low dietary vitamin A for more than 168 d but not for less than 145 d has decreased both serum and hepatic retinal (Gorocica-Buenfil et al., 2007a,b,c). Results in this experiment agree with our previous findings. If vitamin A inhibits i.m. fat deposition, it remains unknown how low serum retinol levels need to be to observe benefits in marbling score. Furthermore, it is still not known how long serum retinol levels need to be reduced to stimulate adipocyte differentiation in the i.m. depot. Further research is needed to improve our understanding on the retinol depletion kinetics in the liver.
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; Adachi et al., 1999). In the present experiment, there was no relationship (P > 0.71) between marbling score and serum or liver retinol. Previously, we have reported a significant negative correlation between marbling score and serum and liver retinol at slaughter (Gorocica-Buenfil et al., 2007a,b). The lack of significance between marbling score and retinol levels in the present experiment provides further evidence that other factors (such as roasted soybean inclusion or energy restriction) may have attenuated the marbling response to dietary vitamin A restriction.
The effect of dietary vitamin A restriction on s.c. adipose fatty acid composition is presented in Table 5. Vitamin A and its precursor β-carotene have been reported to reduce SCD activity (Alam and Alam, 1985; Siebert et al., 2003). This enzyme catalyzes the endogenous synthesis of CLA, and this is the main source of CLA in ruminant tissues (Madron et al., 2002; Palmquist et al., 2004). Thus, feeding low vitamin A diets may attenuate this reduction in SCD activity. Increasing SCD activity may result in increased CLA levels in beef tissues, because more rumenic acid could be synthesized from vaccenic acid (Griinari and Bauman, 1999). Changes in SCD activity would also result in more MUFA and PUFA and a lower proportion of SFA. In this experiment, dietary vitamin A restriction did not increase rumenic or total CLA content in s.c. fat (Table 5). This is in agreement with our previous experiments (Gorocica-Buenfil et al., 2007a,b,c). Thus, it can be concluded that feeding low vitamin A diets does not appear to be a suitable strategy to increase CLA in beef tissues.
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; Smith et al., 2002). These indicators suggest that SCD activity was slightly affected by dietary vitamin A restriction. Conflicting reports are present in the literature regarding the effect of retinol on SCD activity. Some authors suggest that retinol and β-carotene reduce SCD activity (Alam and Alam, 1985; Siebert and Zurk, 2004), whereas others report that retinol inhibits SCD transcription (Daniel et al., 2004) resulting in increased levels of CLA in human tissues of patients with high vitamin A plasma levels (Lucchi et al., 2005). Results in this experiment may suggest that SCD activity was increased by dietary vitamin A restriction. However, changes in fatty acid composition, although significant, were not large enough to consider this a practical strategy to modify fatty acid composition of beef tissues.
Feeding low vitamin A diets may be an effective way to increase marbling without affecting s.c. deposition. However, it would be unethical to increase marbling if this practice was detrimental to animal health and welfare. This is the fourth experiment we have conducted evaluating vitamin A restriction, and we have not observed a single case of clinical or subclinical vitamin A deficiency. An objective of this experiment was to evaluate the immunocompetency of vitamin A-restricted steers late in the feeding period. Maintaining the immune system and specifically supporting adequate immunoglobulin G production is one of the many important functions of vitamin A (Semba, 1998). Vitamin A-restricted rats showed a severe reduction in antibody production after an immune challenge even before clinical symptoms of vitamin A deficiency were observed (Pasatiempo et al., 1990). Thus, to evaluate the immunocompetency of vitamin A-restricted steers, the antibody response to an ovalbumen vaccine was measured late in the feeding period. Ovalbumin titer was not affected by vitamin A restriction (LOW = 13.1 vs. HIGH = 12.8 log2 titers, P = 0.60). These data taken together with the performance data and the results of our previous experiments clearly suggest that the NRC recommendation for vitamin A in growing cattle should be revised. NRC (1996) recommendations are based on sparse information published over 35 yr ago (Perry et al., 1965, 1968; Eaton et al., 1972). Evaluating the effect of vitamin A at concentrations below the NRC recommendation on the immune response to production-relevant antigens is warranted. Before adjusting the dietary vitamin A level to modulate the site of fat deposition, nutritionists require convincing evidence that the effects of this management strategy do not affect animal health and well-being. Further research is needed to determine the vitamin A requirement of growing cattle and to identify potential nutrient interactions that would affect this requirement.
In conclusion, feeding a low vitamin A diet for 216 d to Angus-based steers did not affect performance, marbling score, or measures of animal health or immunocompetency. These steers were fed 20% roasted soybeans and were limit-fed for 141 d, which may have attenuated the vitamin A response. Slight changes in the fatty acid profile were observed, suggesting that vitamin A restriction may have affected desaturase enzyme activity.
1 Corresponding author: loerch.1{at}osu.edu
Received for publication April 26, 2007. Accepted for publication March 10, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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9-desaturase in the production of cis-9, trans-11 CLA. J. Nutr. Biochem. 12:622–630.[CrossRef][Medline]9 desaturase index and stearoyl coenzyme A desaturase enzyme activity in porcine subcutaneous adipose tissue. J. Anim. Sci. 80:2110–2115.This article has been cited by other articles:
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  G. J. Hausman, M. V. Dodson, K. Ajuwon, M. Azain, K. M. Barnes, L. L. Guan, Z. Jiang, S. P. Poulos, R. D. Sainz, S. Smith, et al. BOARD-INVITED REVIEW: The biology and regulation of preadipocytes and adipocytes in meat animals J Anim Sci, April 1, 2009; 87(4): 1218 - 1246. [Abstract] [Full Text] [PDF]
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S. B. Smith, H. Kawachi, C. B. Choi, C. W. Choi, G. Wu, and J. E. Sawyer Cellular regulation of bovine intramuscular adipose tissue development and composition J Anim Sci, April 1, 2009; 87(14_suppl): E72 - E82. [Abstract] [Full Text] [PDF]
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