If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Vitamin E comprises 8 fat-soluble isoforms: α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol. Yet the body preferentially uses α-tocopherol, and only α-tocopherol supplementation can reverse vitamin E deficiency symptoms. However, other isoforms influence many biological functions in the body, including inflammation and stress. Therefore, the study objective was to determine metabolic and performance responses in young calves fed diets containing a constant amount of α-tocopherol and increasing amounts of soybean oil-derived mixed γ- and δ-tocopherols. Holstein calves [n = 48; 2–3 d of age; 40.2 kg of initial body weight (BW), standard error = 0.54] were assigned to receive approximately 0, 5, 10, or 15 mg/kg of BW daily (treatments T0, T1, T2, and T3, respectively) of mixed tocopherols (TMIX) provided in milk replacer (MR) and calf starter. The TMIX liquid contained 86% γδ-tocopherols and 9% α-tocopherol. Milk replacers were formulated to contain approximately 0, 400, 800, or 1,200 mg of TMIX/kg for treatments T0, T1, T2, and T3, respectively. Calf starters were formulated to contain approximately 0, 250, 500, or 750 mg of TMIX/kg for treatments T0, T1, T2, and T3, respectively. Mean consumption of γδ-tocopherols was 0.0, 6.5, 14.3, and 20.5 mg/kg of BW, respectively. Milk replacer contained 24% crude protein (CP) and 20% fat on a dry matter (DM) basis. Calf starters were pelleted and offered for ad libitum consumption from 0 to 56 d. Starters contained 18 to 20% CP and 9 to 12% starch in the DM. On d 28, 4 calves per treatment were randomly selected for slaughter, and necropsy was performed. Samples of liver, duodenum, ileum, and trapezius muscle were collected and stored before analysis for α-, β-, γ-, and δ-tocopherols and δ-tocotrienol. Data were analyzed using a completely randomized design using mixed model ANOVA with orthogonal polynomials to determine linear and quadratic effects of TMIX. Repeated-measures analyses were performed for data collected over time. Increasing dietary TMIX increased or tended to increase change in hip width at 28 and 56 d, respectively, and improved average daily BW gain and gain-to-feed ratio at 56 d. Increasing TMIX reduced plasma xanthine oxidase at 0 h and tended to reduce concentrations at 24 h following vaccination with 2 commercial vaccines on d 28; however, we detected no effect of TMIX following vaccination on d 56. Concentration of α-tocopherol in skeletal muscle declined quadratically with increasing TMIX, whereas ileal and liver γ-tocopherol increased linearly with increasing TMIX. The number of mucin-2 cells in the ileum increased more than 2-fold in calves fed T3. Addition of mixed tocopherols to diets of young dairy calves improved animal growth and altered indices of antioxidant metabolism.
Nutrient requirements for water- and fat-soluble vitamins are often determined by measuring the amount of a vitamin required to eliminate signs of deficiency (
). On the other hand, consumption of greater vitamin amounts may influence productive responses, such as growth or immunity. The 2001 NRC committee increased the requirement for vitamin E from 40 to 50 IU/kg of DM in dairy calf milk replacers because the additional 10 IU/kg of DM might augment the immune system of calves under stress (
). Therefore, determination of optimal inclusion of a vitamin in production diets depends on whether the goal is elimination of clinical deficiency signs or positively influencing growth or immunity, or both.
Recommendations for dietary vitamin E in young calves vary. For example, the 2001 Dairy NRC (
) recommended that vitamin E be 2.4 to 3.4 IU/kg of BW (125 to 250 IU) per day. These authors reported that serum α-tocopherol concentration increased linearly at 125 or 250 IU of dietary vitamin E per day, but the increase in serum α-tocopherol concentration was smaller when supplementation was increased from 250 to 500 IU/d. Serum concentrations also tended to decline over time and with increasing age, BW, and intake. The authors concluded that a constant amount of vitamin E provided daily to 24 wk of age did not maintain constant serum α-tocopherol concentration, and they recommended that the best approach to ensure satisfactory circulating vitamin E is to base allowances on BW. More recent studies suggest a role of α-tocopherol alone or in combination with other fat-soluble vitamins in acute infection (
Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.
reported that, for every 100-IU increase in vitamin E intake, there was a 0.35% reduction in morbidity in beef calves received at a feedlot without effect on growth, intake, or feed efficiency. Others have reported that vitamin E supplementation improves immune response to Pasteurella multocida vaccination in crossbred calves (
reported an association between BW gain and vitamin E status, suggesting a need for increased vitamin E in milk replacers fed to calves managed for increased rates of gain.
The term vitamin E commonly refers to α-tocopherol, but actually encompasses α-, β-, γ-, and δ-isoforms of tocopherol as well as α-, β-, γ-, and δ-isoforms of tocotrienols (
). Although the α-isoform is usually considered the most biologically active and important, recent research suggests that other isoforms of vitamin E may have essential roles in health, reproduction, and growth. For example,
reported that, whereas α-tocopherol has been shown to exhibit anti-inflammatory properties in many species, γ-tocopherol is proinflammatory. Others have shown that α- and γ-tocopherols may be complementary in responses to reducing effects of oxidation and nitration in different types of cells (
Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo.
). Few studies have documented the value of supplementation of calves with isoforms of tocopherol other than α-tocopherol. Therefore, our objective was to determine effects of additional mixed tocopherols on animal performance and health, and selected indices of tocopherol status, antioxidant status, and immune response. Our null hypothesis was that increasing amounts of γ-tocopherols at fixed concentration of α-tocopherol would have no effect on intake, growth, and indices of antioxidant status or immune response.
MATERIALS AND METHODS
All animals were cared for as described in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (
) and under approval of the Institutional Animal Care and Use Committee (Cargill Animal Nutrition) for protocol F4-C1706. The trial was conducted between July 27 and September 20, 2017.
Animals and Experimental Design
Power analysis was conducted using power (1 − β) = 80%, and α-level = 0.05. We chose daily BW gain as performance parameter and based power analysis on variation in published studies from our group using similar facilities and protocols (
Effect of milk replacer feeding rate, age at weaning, and method of reducing milk replacer to weaning on digestion, performance, rumination, and activity in dairy calves to 4 months of age.
). Using a minimum difference of 100 g/d for daily BW gain, the minimal sample size needed was 8 calves per treatment.
Holstein calves (n = 48, 1 female, 47 males; 40.2 kg initial BW, SE = 0.54) were born at a single dairy farm and fed 1.8 L of pooled colostrum 3 times (approximately 2, 8, and 14 h of age) in the first 24 h. Thereafter, they were fed milk replacer (MR; 1.8 L per feeding, twice daily) until they were transported 3.5 h to the experimental site (Nurture Research Center, Provimi North America, New Paris, OH). Calves were 2 to 3 d of age at study initiation. Calves were weighed on the day after arrival, blood was collected by jugular venipuncture, and serum was separated by centrifugation at 3,000 × g at room temperature for 15 min (VWR International). Total serum protein concentration was estimated using an optical refractometer (ATAGO USA Inc.). Calves were weighed, hip height and widths were measured, and then calves were assigned randomly to receive 1 of 4 experimental diets, formulated to provide approximately 0, 5, 10, or 15 mg/kg of BW of mixed γδ-tocopherols (TMIX) daily (T0, T1, T2, and T3, respectively).
Feeding and Housing
Concentrated mixed tocopherol oil was extracted from soybeans using a proprietary technology (Cargill Innovation Center, Velddriel, the Netherlands). The TMIX contained 86% γδ-tocopherols and 9% α-tocopherol.
Milk replacers were formulated to contain 144 IU of α-tocopherol/kg (as-fed basis) and 0, 400, 800, or 1,200 mg of TMIX/kg (as-fed basis) for treatments T0, T1, T2, and T3, respectively. Concentration of α-tocopherol in basal MR provided 36 IU of vitamin E/kg (as-fed basis). Supplemental α-tocopherol and mixed tocopherols were provided by 4 additives for MR (Table 1) that were added to reconstituted MR at each feeding at 5 g/calf. Milk replacer was a 24% CP, 20% fat all-milk protein formula containing whey protein concentrate as primary protein source and lard as primary fat source. The same MR was used for all treatments.
Table 1Formulation of milk replacer mixed tocopherol (TMIX) additives
Calf starters (CS; Table 2) were formulated to contain 90, 84, 78, and 72 IU/kg (as-fed basis) of α-tocopherol and 0, 250, 500, and 750 mg of TMIX/kg for treatments T0, T1, T2, and T3, respectively. All CS were pelleted to approximately 5-mm diameter pellets. Basal CS vitamin and mineral premix (Table 2) provided vitamins (except vitamin E) and minerals to meet or exceed NRC requirements (
). Separate vitamin E and TMIX premixes were added to CS to provide approximately equal amounts of α-tocopherol and graded levels of TMIX, respectively, in each diet. Calculated contributions of α-tocopherol and γδ-tocopherols in CS and MR are shown in Table 3.
Additives included base vitamin and mineral premix (0.75%), monocalcium phosphate (0.68%), salt (0.6%), decoquinate premix (Zoetis, 0.5%), calcium carbonate (0.48%), binder (0.25%), and diflubenzuron premix (Clarifly, 0.07%; Central Life Sciences).
1 Treatments: T0, T1, T2, and T3 formulated to provide 0, 5, 10, and 15 g of γδ-tocopherols per kg of BW.
2 Additives included base vitamin and mineral premix (0.75%), monocalcium phosphate (0.68%), salt (0.6%), decoquinate premix (Zoetis, 0.5%), calcium carbonate (0.48%), binder (0.25%), and diflubenzuron premix (Clarifly, 0.07%; Central Life Sciences).
3 Vitamin E premix contained 1.2% Vitamin E 50 (DSM Nutritional Products) and 98.8% wheat middlings.
Contribution of tocopherols from feed was calculated using estimated DM intake from calf starter and milk replacer. Milk replacer intake was estimated as 640 g of DM/d from wk 1 to 5, 500 g/d during wk 6, and 0 g/d thereafter. Calf starter intake was estimated using the equation calf starter DMI (g/d) = 150.9 − 180.1 × age (wk) + 50.1 × age2.
2 Contribution of tocopherols from feed was calculated using estimated DM intake from calf starter and milk replacer. Milk replacer intake was estimated as 640 g of DM/d from wk 1 to 5, 500 g/d during wk 6, and 0 g/d thereafter. Calf starter intake was estimated using the equation calf starter DMI (g/d) = 150.9 − 180.1 × age (wk) + 50.1 × age2.
3 Treatments: T0, T1, T2, and T3 formulated to provide 0, 5, 10, and 15 g of γδ-tocopherols per kg of BW.
All calves were fed 0.66 kg of DM/d of MR (24% CP, 19.9% fat) to 39 d, then 0.33 kg of DM/d to weaning at 42 d. The MR was reconstituted to 14% solids with water (45°C) and fed at approximately 0600 and 1600 h to d 39, then at 0600 h from d 40 to 42 via buckets with nipples. Calves were offered CS without added forage to d 56. Amount of feed offered and previous day's feed refusals were weighed once daily to determine daily DM intake. Starters were offered once daily at approximately 0800 h. Water was offered for ad libitum consumption at approximately 0800 h. Water consumption was not measured.
Calves were housed in a curtain-sided, naturally ventilated nursery with no added heat in 1.2- × 2.4-m individual pens bedded with wheat straw. Dividers were metal fence panels, which allowed individual calves to make contact with each other and visually interact. Treatments were randomly assigned within the barn. Every other bag of starter and every bale of hay was sampled and composited for nutrient analysis.
Measurements and Analyses
Calves were weighed initially and every 7 d thereafter to 56 d. Hip widths were measured with a caliper, and BCS was estimated initially and every 14 to 56 d. Body condition score was based on a 1-to-5 system using 0.25-unit increments, with 1 being emaciated and 5 being obese (
), based on changes around the vertical and transverse processes of the spine as palpated by one experienced technician. Feces were scored daily on a scale of 1 = firm, normal, 2 = less firm, normal, 3 = thick, batter-like, 4 = thin, batter-like, 5 = watery; modified from
. Intermediate scores of 1.5, 2.5, 3.5, and 4.5 were recorded as appropriate. An abnormal fecal day was recorded when fecal score was ≥3. A medical day was recorded when a calf was treated, according to predefined protocol, with an antibiotic or anti-inflammatory medication. Composites of feeds, refused feed, and feces were analyzed for DM (oven method 930.15; AOAC International, 2000), ash (oven method 942.05; AOAC International, 2000), CP (Kjeldahl method 988.05; AOAC International, 2000), fat ether extract (alkaline treatment with Röse-Gottlieb method 932.06 for milk powder; diethyl ether extraction method 2003.05 for CS and hay;
Symposium: Carbohydrate methodology, metabolism and nutritional implications in dairy cattle. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
Determination of starch, including maltooligosaccharides, in animal feeds: A comparison of methods and a method recommended for AOAC collaborative study.
On d 28 at approximately 1100 h, 4 calves on each treatment were randomly selected and transported by truck approximately 1.5 h to the Ohio State University Department of Animal Science (Columbus, OH). Calves were fed their respective afternoon feeding at approximately 1600 h. Beginning at 0600 h on d 29, calves were stunned with a captive bolt, followed by exsanguination. Digestive organs were exteriorized, and samples of liver, duodenum, and ileum and a sample of trapezius muscle were collected and placed in formalin solution before analysis for α-, β-, γ-, and δ-tocopherols and δ-tocotrienol as defined by
The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows.
Measurement of ileal morphology and relative tissue content of mucin-2 cells (MUC-2) were obtained by microscopy and quantitative image analysis of dual color immunofluorescent slides, respectively, as described by
. In brief, tissues were fixed in 4% paraformaldehyde and preserved in ethanol and further embedded in paraffin, sectioned at 5 µm, and mounted on glass slides (Histoserv Laboratory Inc.). After standard specimen preparation for immunofluorescence, measurements of villus length and crypt depth were made using the image layer associated with the blue channel of the immunofluorescence wherein nuclei were visualized by 4′,6-diamidino-2-phenylindole staining. The MUC-2 were visualized in the specimens using rabbit polyclonal anti-MUC2 antibody as the primary antibody (Novus USA Inc.) and, as the reporter, goat-anti-rabbit IgG conjugated with near-infrared Alexa 647 to minimize autofluorescence (Thermo Fisher Scientific). Tissue sections were photographed under a 4× objective using an Olympus IX-73 microscope (Olympus America Inc.) equipped with a Hamamatsu Orca 2 Deep Cooled CCD high-resolution digital camera (Hamamatsu Ltd.) with the Alexa 647 channel layer pseudocolored red. Captured raw images were composited into a master image sheet, which was then opened in Image-Pro Premier Version 9.3 (Media Cybernetics Inc.). In this manner, any change in the visualization-pixel recognition algorithm was applied across all images simultaneously. The areas of interest for quantifying the pixel density specific to MUC-2 in the specimens were manually outlined and localized to the villi and crypts. Nonspecific Alexa 647 pixilation was conservatively defined as any pixel cluster less than 5 × 5 pixels via gating, and these clusters eliminated from the pixel summation. For normalization, total MUC-2 pixels per nucleus were statistically used for the treatment comparisons.
On d 28 and 56, rectal temperatures and jugular blood samples were collected at 0730 h from calves not selected for necropsy. At 0800 h, all calves were vaccinated intramuscularly with Presponse HM (Pasteurella multocida bacterial extract–Mannheimia haemolytica toxoid, Boehringer Ingelheim) and Bovi-Shield Gold 5 (bovine rhinotracheitis virus diarrhea parainfluenza3-respiratory syncytial virus vaccine, Zoetis Inc.). Rectal temperatures were measured at 0945, 1145, and 1545 h, and 0800 h the following day. Additional jugular blood samples were collected at 1000 h (2 h after vaccination), 1200 h (4 h), and 1600 h (8 h), and 0800 h the following day (24 h). Jugular blood samples were collected into evacuated tubes containing EDTA and centrifuged at 3,000 × g for 15 min at room temperature. Plasma was removed and placed into sample vials which were frozen at −20°C.
Plasma samples collected at −0.5 and 24 h after vaccination on d 28 and 56 were analyzed for xanthine oxidase (XO;
), and virus neutralization titers to bovine viral diarrhea virus (BVD) and parainfluenza 3 (PI3; Ohio Department of Agriculture Animal Disease Diagnostic Laboratory, Reynoldsburg, OH). Plasma samples collected on d 28 and 56 at −0.5, 2, 4, 8, and 24 h after vaccination were analyzed for TNF-α concentrations (
Data were analyzed as a completely randomized design with experimental treatment (level of TMIX in the diet) as fixed effect and calf as random effect. The Mixed procedure of SAS (version 9.4, SAS Institute Inc.) was used. Daily observations were averaged by week, and repeated measures were incorporated into models when variables were measured over time. An autoregressive type 1 covariance matrix was employed, as determined using Akaike's information criteria for most performance variables. Uncorrelated covariance matrix improved model fit for TNF-α and rectal temperature data. Fecal scores were analyzed using Proc GLIMMIX with repeated measures. Fixed effects included treatment, week, and treatment × week interaction. Calf was included as a random effect. Normality distribution was assessed by the Shapiro-Wilk test, and homogeneity of the variances using the Levene test. Orthogonal polynomial contrast statements were constructed to evaluate effects of 0, 5, 10, or 15 mg of TMIX/kg of BW; differences were declared at P ≤ 0.05 and tendencies at P ≤ 0.10. Performance data were analyzed separately for 0 to 4 wk and 5 to 8 wk because of different numbers of observations during each period.
RESULTS
Mean temperature inside the barn during the trial was 21.5°C (SD = 7.0) and ranged from 6.8 to 39.3°C. Chemical compositions of diets used in the trial are shown in Table 4. Least squares means of animal performance during the first and second 4 wk of the trial are presented in Table 5, Table 6. We found few differences in performance measures during the first 4 wk of the study (Table 5), except changes in hip width, which increased linearly with increasing TMIX intake. Intake of α-tocopherol was 2.3, 2.1, 2.3, and 2.2 IU/kg of BW and intake of γδ-tocopherol was 0, 6, 13, and 19 mg/kg of BW for T0, T1, T2, and T3, respectively, over the first 4 wk of the study.
1 Treatments: T0, T1, T2, and T3 formulated to provide 0, 5, 10, and 15 g of γδ-tocopherols per kg of BW; n = 12 observations per treatment.
2 F-values for main effects.
3 Contrasts: L = linear, Q = quadratic.
4 Total change in BCS over the 56-d period. BCS was based on a 1-to-5 scale using 0.25-unit increments, with 1 being emaciated and 5 being obese.
5 Where 1 = normal, thick in consistency; 2 = normal but less thick; 3 = abnormally thin but not watery; 4 = watery; 5 = watery with abnormal coloring.
1 Treatments: T0, T1, T2, and T3 formulated to provide 0, 5, 10, and 15 g of γδ-tocopherols per kg of BW; n = 8 observations per treatment.
2 F-values for main effects.
3 Contrasts: L = linear, Q = quadratic.
4 Total change in BCS over the 56-d period. BCS was based on a 1-to-5 scale using 0.25-unit increments, with 1 being emaciated and 5 being obese.
5 Where 1 = normal, thick in consistency; 2 = normal but less thick; 3 = abnormally thin but not watery; 4 = watery; 5 = watery with abnormal coloring.
During wk 5 to 8, BW gain, gain-to-feed ratio, and final hip width increased linearly with increasing TMIX, and change in hip width tended to increase linearly (Table 6). Consumption of γδ-tocopherol was greater per unit of BW during wk 5 to 8 compared with wk 0 to 4 and were 0, 7, 16, and 22 mg/kg of BW for T0, T1, T2, and T3, respectively.
Least squares means of weekly CS intake during wk 1 to 4 (Figure 1) were affected by a week × treatment interaction. From wk 3, calves fed T2 and T3 consumed or tended to consume more calf starter DM compared with other treatments. However, from wk 5 to 8, no significant effects of treatment or week × treatment on CS intake were detected (data not shown).
Figure 1Least squares means of starter DM intake in calves (n = 12/treatment) fed diets formulated to provide 0, 5, 10, or 15 mg/kg of BW of γδ-tocopherols daily (T0, T1, T2, and T3, respectively). Significant effects of week (P < 0.001) and week × treatment (P = 0.03). Error bars indicate SEM (0.015).
Plasma concentrations of XO (Table 7) at −0.5 h after vaccination on d 28 were affected by linear and quadratic effects of TMIX. Calves fed TMIX at any concentration had consistently lower XO concentrations compared with the negative control before vaccination. Similarly, at 24 h following vaccination on d 28, XO concentrations tended (P = 0.07) to decline linearly with increasing TMIX. However, changes in concentrations from −0.5 to 24 h after vaccination were unaffected by dietary treatment. At 56 d, XO concentrations at −0.5 and 24 h and the change in concentration following vaccination were unaffected by treatment.
Table 7Least squares means of selected plasma metabolite concentrations of calves 30 min before (−0.5 h) and 24 h after (24 h) vaccination on d 28 and 56
Plasma urea N concentrations at −0.5 and 24 h after vaccination on d 28 and 56 were unaffected by TMIX and averaged 4.4, 8.5, 11.1, and 11.2 mmol/L, respectively.
Haptoglobin concentrations at 28 d did not differ from zero in calves fed T0, T1, or T2 at −0.5 h following vaccination, but were elevated in calves fed T3. By 24 h after vaccination, haptoglobin concentrations increased in calves fed T0, T1, and T2 and did not change in calves fed T3. Similarly, low concentrations of haptoglobin increased following vaccination with no effect of TMIX at 56 d.
Titers to BVD at 28 d declined linearly with increasing TMIX in the diet (Table 7) in plasma collected before vaccination, although no treatment effect was observed at 56 d. Titers to PI3 were unaffected by treatment at either 28 or 56 d.
Tissue concentrations of α-, γ-, and δ-tocopherols (Table 8) collected from calves on d 29 were variably affected by treatment. Concentrations of α-tocopherol were highest in liver and ileum, without effect of TMIX addition. Skeletal muscle α-tocopherol declined quadratically from 6.55 to 2.43 µg/g of tissue in calves fed T0 to T2, respectively. Concentrations increased in calves fed T3.
Concentrations of β-tocopherol and δ-tocotrienol were below minimum detectable concentration (<0.5 μg/g). Concentrations of α-, β-, and γ-tocotrienol were not measured.
and ileal morphology in calves collected on d 29 of the study
1 Concentrations of β-tocopherol and δ-tocotrienol were below minimum detectable concentration (<0.5 μg/g). Concentrations of α-, β-, and γ-tocotrienol were not measured.
2 Treatments: T0, T1, T2, and T3 formulated to provide 0, 5, 10, and 15 g of γδ-tocopherols per kg of BW; n = 4 calves per treatment.
Concentration of γ-tocopherol in ileum and liver increased linearly with increasing TMIX. We detected no significant effects of TMIX in the duodenum, and concentrations were below minimum detectable limits in muscle. Concentrations of δ-tocopherol were unaffected by treatment and were highest in duodenal and ileal tissue and lower in liver.
Addition of TMIX to the diet had no effect on gross ileal villus height or crypt depth (Table 8); however, mean MUC-2 pixels per expressing cell increased quadratically with increasing TMIX. As can be seen in Figure 2, calves fed more TMIX had more MUC-2 both in the goblet mucin-secreting cells and in the mucus layer covering the epithelial surface. Concentrations in calves fed T3 were 2-fold higher than control, although differences among other treatments were minimal.
Figure 2Representative visualization of ileal nuclei (blue channel), mucin-2 (MUC-2) cells (red channel), the relationship of immuno-MUC-2 staining relative to nuclear localization (overlay), and image analysis of MUC-2-specific pixel identification (yellow pixels) in tissue samples from a control calf (0 dietary added mixed tocopherols, TMIX) and a calf fed 15 mg/kg of BW of TMIX.
Least squares means of plasma TNF-α concentrations on d 28 (Table 9) were elevated at −0.5 and 2 h after vaccination and then declined to <9 pg/mL by 24 h after vaccination. We detected no effect of TMIX on plasma TNF-α at 28 d. At 56 d, plasma TNF-α concentrations did not change markedly from −0.5 to 8 h but then increased to 24 h. Across all time periods, concentrations of plasma TNF-α tended (P < 0.10) to be lower in calves fed T1 and T2 compared with T0 and T3.
Table 9Least squares means of plasma TNF-α concentration and rectal temperatures of calves following vaccination
Rectal temperatures on d 28 and 56 followed a similar pattern following vaccination (Table 9). Temperatures increased until approximately 8 h after vaccination and then declined to 24 h following vaccination. No effect of TMIX addition on rectal temperatures was detectable.
DISCUSSION
An important objective of our study was to determine absorption and sites of tissue storage of γ- and δ-tocopherols in calves fed TMIX. Concentrations of α-tocopherol were unaffected by TMIX in duodenum, ileum, and liver samples, although muscle concentrations were affected quadratically by TMIX inclusion (Table 9). Concentrations were lowest in muscle, which is consistent with reports in adult cattle (
Concentrations of γ-tocopherol increased with increasing TMIX in the ileum but not in the duodenum, which is consistent with the theory that vitamin E isoforms are absorbed mainly in the distal intestine (
). Highest concentrations of δ-tocopherol were found in the duodenum and ileum. That concentrations >10 µg/g of wet tissue were found in the duodenum suggests that at least some δ-tocopherol may be absorbed in the proximal intestine.
Liver γ-tocopherol increased with increasing TMIX, indicating absorption and storage of this isoform.
Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo.
reported that liver concentrations of γ-tocopherol increased in calves fed γ-tocopherol for 5 d, consistent with our observation. Conversely, liver δ-tocopherol concentrations were similar to basal levels of γ-tocopherol and were refractory to dietary supplementation. Concentrations of γ- and δ-tocopherol were not measurable in muscle, which is consistent with a lack of response to dietary γδ-tocopherol in rats (
reported increasing liver stores of α- and γ-tocopherols with supplementation of the respective isoform in MR diets of Holstein calves. Calves (12 d of age) were fed 2.2 or 6.6 mg/kg of BW of α- or γ-tocopherol in MR for 14 d. Increasing α- and γ-tocopherol increased liver concentrations by 31 and 96%, respectively. These authors also reported that supplementation with γ-tocopherol (and similar α-tocopherol intake) increased liver and heart muscle concentrations of α-tocopherol, and they suggested that γ-tocopherol may be used preferentially as an antioxidant in vivo, sparing α-tocopherol, which is then stored in tissues. This effect on tissue concentrations of α-tocopherol was also observed in rats fed a constant amount of α-tocopherol and graded amounts of γ-tocopherol (
Graded dietary levels of RRR-γ-tocopherol induce a marked increase in the concentrations of α- and γ-tocopherol in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats.
). However, we did not observe a similar response. Increasing dietary TMIX did not affect tissue concentrations of α-tocopherol in the intestine or liver, and skeletal muscle concentrations declined quadratically with increasing TMIX.
The effects of short-term feeding of tocopherol mix (α-, β-, γ-, and δ) on blood neutrophil function and immunometabolic-related gene expression in lactating dairy cows.
reported that feeding TMIX increased γ-tocopherol concentrations but not α-tocopherol concentration in liver, mammary gland, and muscle tissues from lactating cows. The liver had the highest ability to store α- and γ-tocopherol. These results are generally consistent with data from our study, although calves in our study stored γ-tocopherol in the ileum, and we found no γ-tocopherol in calf muscle.
Reactive oxygen species such as superoxide anion (O2•−) and reactive nitrogen species such as peroxynitrate anion (ONOO−) may damage tissues and overwhelm the body's endogenous antioxidant capacity. Antioxidative enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase, are essential components of oxidative stress defense in animals, including calves, and function to neutralize toxic reactants by changing their chemical reactivity. Other strategies that counter the reaction effects of these chemical adducts include the availability of antioxidant metabolites such as α- and γ-tocopherols, which serve as targets for the oxidative and nitrative reactants that would otherwise chemically modify cellular components, causing cell damage.
), and it may be important to neonatal calf health. Oxidative status plays a key role in initiation and maintenance of diseases such as diarrhea and pneumonia in calves (
). Dietary antioxidants play an important role in maintaining oxidative stress, and provision of adequate amounts of dietary antioxidants is important to calf health.
Nitration reactions may also influence animal metabolism, growth, and efficiency. Reactive nitrogen species such as nitric oxide and ONOO− (derived when NO reacts with O2•−) may interact with tyrosine residues in proteins, causing nitration of the phenolic ring in the 3′-position and a change in protein structure, and, possibly, in function. The presence of tyrosine-nitrated proteins indicates nitro-oxidative stress (
Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo.
reported increased in nitro-oxidative damage (measured as nitrotyrosine immunostaining) when young calves were challenged with Eimeria bovis. γ-Tocopherol is a competitive target for nitration of protein tyrosine and is capable of mitigating nitration, thereby maintaining normal protein function (
). Calves fed TMIX in our study had greater concentrations of γ-tocopherols in liver and ileum (Table 8), suggesting a more robust defense against protein nitration. Although ileal morphology was unaffected by increasing TMIX in the diet (Table 8), the number of MUC-2-producing cells was highest in calves fed T3. Mucin is an important defense against pathogens (
The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.
), and increased levels of immunoreactive MUC-2, a marker for mucin presence, could reflect a heightened barrier against enteric infection or the inward leakage of gut endotoxin in TMIX-fed animals. Immunofluorescence data (Figure 2) suggest that distribution patterns for MUC-2 were affected by TMIX, as evidenced by a greater abundance surrounding the villi at the lumen-villus border, presumably an increased amount of mucin protecting the integrity of the epithelial surface.
Body weight gain, change in hip width, and efficiency of BW gain were improved from wk 5 to 8 of our study with increasing TMIX supplementation. Differences in growth were not mediated through reduced days with diarrhea, as number of antibiotic treatments and number of abnormal fecal days were unaffected by treatment. Generally, calves were healthy and required few treatments or experienced few abnormal fecal days. However, improvement of oxidative status, particularly early in life when oxidative homeostasis is incompletely established, and reduction of protein tyrosine nitration with TMIX addition may be responsible for additional nutrients available for growth. Improved feed efficiency in our calves from wk 5 to 8 suggests this is a possible mode of action of TMIX.
Starter CP was numerically higher in T1, T2, and T3 diets compared with T0 (Table 4). It is unclear whether differences in calf starter CP may have influenced BW gain and change in hip width from 5 to 8 wk of age. Using diets and feeding programs similar to those used in this study,
also reported that feeding CS containing 26% CP tended to increase mass of reticulorumen and liver compared with a CS containing 21.5% CP.
Concentrations of XO on d 28 (Table 7) were lower in calves fed TMIX. At 24 h following vaccination, concentrations of XO increased in all calves but tended to (P < 0.07) decline linearly with increasing TMIX. Plasma XO is a marker of cellular leakage of an enzyme capable of generating reactive oxygen intermediates such as O2•−. Thus, lower plasma XO at 28 d suggests improved oxidative status and greater resistance to oxidative challenge. Concentrations of plasma XO were similar to those of
, who reported that basal concentrations of XO were <5 mU/mL of plasma in older beef heifers (>400 kg of BW) but increased to 9 to 14 mU/mL following LPS challenge. Also, addition of mixed tocopherols to the diet of heifers reduced the increase in plasma XO concentrations following LPS challenge (
Plasma XO and PUN increased following vaccination were greater at 28 d compared with 56 d (Table 7), suggesting that the ability of the calf to respond to vaccination may be influenced by age. Indices of antioxidant status improve with age in newborn calves, and effects of protein oxidation and advanced oxidation protein products (indices of protein tyrosine nitration) decline with advancing age in calves (
reported that oxidative status of calves improved 2 h after ingestion of colostrum, and suggested that improved status may be due to transfer of antioxidants in colostrum or activation of antioxidative pathways, or both. Also, milk replacers may have lower antioxidant capacity than whole milk (
). Smaller changes in concentration of XO and PUN following vaccination may be associated with improved oxidative status due to maturation of the antioxidant system and weaning in our calves.
Plasma urea N concentrations were unaffected by TMIX inclusion in the diet (Table 8). On d 28, vaccination prompted an increase in BUN concentrations nearly 2-fold, indicating that vaccination stimulated protein catabolism. However, at d 56, concentrations were affected by neither TMIX nor vaccination. Concentrations of PUN at 56 d were >10 mmol/L, higher than in other reports in the literature in calves of similar ages (
Nitrogen utilization, preweaning nutrient digestibility, and growth effects of Holstein dairy calves fed 2 amounts of a moderately high protein or conventional milk replacer.
). Rapid rumen fermentation of CS, increased CS intake following weaning, and low starch concentration likely all contributed to high observed PUN.
Plasma haptoglobin concentrations (Table 8) were generally low and unaffected by dietary treatment but increased at 24 h after vaccination on d 28 and 56.
reported haptoglobin concentrations in calves before weaning ranging from 30 to 50 µg/mL; after weaning, concentrations increased to 30 to 90 µg/mL. Pre-vaccination concentrations in this study were much lower than those reported by
; it is unclear whether methodological differences (analyses were performed in different laboratories) or some biological difference could explain the discrepancy. Very low or undetectable (<10 µg/mL) concentrations were reported in healthy Holstein calves from 1 to 11 wk of age (
Effects of ad libitum milk replacer feeding and butyrate supplementation on behavior, immune status, and health of Holstein calves in the postnatal period.
reported serum haptoglobin concentrations in calves from 1 to 8 d of age ranging from 0 to 1,320 µg/mL with a mean of 130 µg/mL (SE = 130).
Kinetics of elaboration of numerous acute phase proteins in calves (i.e., haptoglobin, serum amyloid A, and α-1 acid glycoprotein) differs according to the nature of the perceived stress and type of acute-phase proteins elaborated over time (
, who fed newborn calves no colostrum or colostrum replacer supplemented with vitamin A, D, or E separately, or with A, D, and E combined. Calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Serum haptoglobin was measured at d 0, 1, 7, and 14 d of age. Addition of vitamin A, D, or E alone or in combination did not influence haptoglobin concentration.
Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.
reported increased serum haptoglobin in 4-wk-old, vitamin-replete calves following challenge with BVD virus. Serum vitamin E concentration declined by 82% in the 8 d following challenge, suggesting that the acute phase response contributed to reduced vitamin status.
Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α-tocopherol, performance, haematology and blood chemistry in male Holstein calves.
J. Anim. Physiol. Anim. Nutr. (Berl.).2018; 102 (29905984): 1167-1180
fed 188 or 354 IU of vitamin E per day and vaccinated calves with Shiga toxin–producing Escherichia coli. Vitamin E supplementation increased serum α-tocopherol concentrations but had no effect on hematology or blood chemistry parameters. Vaccination had no effect on serum α-tocopherol and minimal effect on serum biochemical parameters.
Serum BVD and PI3 titers (Table 8) declined linearly with increasing dietary TMIX at 28 d. Other treatment differences were not significant. Mean titers were similar to those reported by
, although those authors reported increased titers in response to inclusion of a functional fatty acid blend in the diet. It is unclear why TMIX would influence only BVD titer and not PI3 titer at 28 d and have no effect on titers at 56 d. That measurable titers to both BVD and PI3 were observed in plasma of calves before vaccination suggests that passive transfer of these antibodies occurred.
Plasma TNF-α concentrations after vaccination on d 28 and 56 (Table 9) followed different patterns. On 28 d, plasma TNF-α concentrations increased to 2 h after vaccination, then declined to 24 h after vaccination. On d 56, concentrations remained low to 8 h and then increased to 24 h. It is possible that calves were naïve to vaccination at 28 d and the vaccine did not prompt a significant inflammatory response. Maternal antibody titers against BVD and PI3 may also have contributed to a lack of inflammatory response (
reported that circulating concentrations of TNF-α in calves from 0 to 8 wk of age were unaffected by amount of MR offered and averaged 135 pg/mL of plasma.
also reported concentrations of TNF-α in older calves (127 kg of BW) that ranged from 72 to 109 pg/mL. Thus, circulating concentrations of TNF-α in calves in this study suggest that calves were exposed to relatively little immune stress. However,
suggested that responsiveness of young calves to dynamic immune challenge with substances such as endotoxin may vary with age. They suggested that younger calves may be relatively refractory to challenge compared with older calves, either because of a lower innate capacity to respond or due to the natural tendency of these responses to be downregulated through the mechanism of tolerance, both of which may be considered survival mechanisms to the deleterious effects of a prolonged proinflammatory response (
Characterization of calves exhibiting a novel inheritable TNF-α hyperresponsiveness to endotoxin: Associations with increased pathophysiological complications.
Rectal temperatures following vaccination at 28 d and 56 d (Table 9) followed a similar pattern of increase following vaccination, to peak at 8 h after vaccination, and then returned to baseline values by 24 h after vaccination. We detected no effect of TMIX on rectal temperatures.
CONCLUSIONS
Addition of mixed tocopherols to MR and CS diets improved calf growth during wk 5 to 8 and altered several indices of inflammatory status (e.g., XO). Generally, addition of TMIX improved antioxidant state of the calf, potentially allowing more nutrients to be directed toward growth. Calves were healthy throughout the study, with minimal health challenges. It is possible that calves exposed to greater immunological challenge could express greater differences in growth with addition of TMIX. Results of this study suggest that feeding up to 21 mg of TMIX/kg of BW improved growth to 56 d.
ACKNOWLEDGMENTS
The authors acknowledge the capable assistance of R. Schlotterbeck and the farm staff at the Nurture Research Center of Provimi North America (Brookville, OH) for assistance with animal care and handling and sample and data collection. The assistance of L. Deikun, G. Schroeder, Y. Sun, P. Piantoni, R. Cramer, and the staff at the Ohio State University Food Science Department (Columbus, OH) with tissue collection is gratefully acknowledged. The HPLC analyses of tocopherols was conducted at Eurofins Craft Technologies (Wilson, NC). This research was wholly funded by Provimi North America, a division of Cargill Animal Nutrition. The authors have not stated any other conflicts of interest.
REFERENCES
Abdala-Valencia H.
Berdnikovs S.
Cook-Mills J.M.
Vitamin E isoforms as modulators of lung inflammation.
Nitrogen utilization, preweaning nutrient digestibility, and growth effects of Holstein dairy calves fed 2 amounts of a moderately high protein or conventional milk replacer.
Graded dietary levels of RRR-γ-tocopherol induce a marked increase in the concentrations of α- and γ-tocopherol in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats.
Effect of milk replacer feeding rate, age at weaning, and method of reducing milk replacer to weaning on digestion, performance, rumination, and activity in dairy calves to 4 months of age.
Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo.
Characterization of calves exhibiting a novel inheritable TNF-α hyperresponsiveness to endotoxin: Associations with increased pathophysiological complications.
Effects of ad libitum milk replacer feeding and butyrate supplementation on behavior, immune status, and health of Holstein calves in the postnatal period.
Determination of starch, including maltooligosaccharides, in animal feeds: A comparison of methods and a method recommended for AOAC collaborative study.
Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.
The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.
The effects of short-term feeding of tocopherol mix (α-, β-, γ-, and δ) on blood neutrophil function and immunometabolic-related gene expression in lactating dairy cows.
The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows.
Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α-tocopherol, performance, haematology and blood chemistry in male Holstein calves.
J. Anim. Physiol. Anim. Nutr. (Berl.).2018; 102 (29905984): 1167-1180
Symposium: Carbohydrate methodology, metabolism and nutritional implications in dairy cattle. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
00702-5&id=gr1.jpg){kind=link}
00702-5&id=gr2.jpg){kind=link}