Interaction of dietary vitamin D3 and sunlight exposure on B. indicus cattle: Animal performance, carcass traits, and meat quality
A.R. Lobo-Jr. a, E.F. Delgado a,⁎, G.B. Mourão a, A.C.M.S. Pedreira a, A. Berndt b, J.J.A.A. Demarchi c
a Departamento de Zootecnia, Escola Superior de Agricultura “Luiz de Queiróz”, Universidade de São Paulo, 13418-900 Piracicaba, SP, Brazil
b Empresa Brasileira de Pesquisa Agropecuária (Embrapa) – Pecuária Sudeste, 13560-970 São Carlos, SP, Brazil
c Centro de Pesquisas em Nutrição Animal e Pastagens, Instituto de Zootecnia, 13460-000 Nova Odessa, SP, Brazil
a b s t r a c t
Attempts to improve beef tenderness through supplementation with dietary vitamin D3 have been challenged by null results and negative impacts on animal performance and carcass traits. Because vitamin D3 is also synthesised by the animal via ultraviolet radiation from sunlight, the effectiveness of supplementation with dietary vitamin D3 may be modulated by the degree of exposure of the animal to sunlight. Hence, this work aimed to verify whether dietary vita- min D3 modifies meat quality without negatively affecting animal performance and carcass traits in B. indicus beef cattle that were either exposed to or protected from natural sunlight. Forty-two (411 ± 38 kg) Nellore-type castrated males were fed a high-concentrate diet for 45 days after assignment to a treatment group. The treatments comprised combinations of three levels of vitamin D3 [ViTD — none (V0) or 2 × 106 IU of vitamin D3 administered for either 2 (V2) or 8 (V8) consecutive days pre-slaughter] and two shading conditions (SHADE — unshaded or shaded). The post-mortem (pm) measurements were taken in the Longissimus thoracis et lumborum muscle. The animal performance and carcass traits were unaffected by ViTD or SHADE. The V2 treatment increased the Myofibrillar Fragmentation Index in shaded animals compared to unshaded ones. Animals under shade had higher muscle calcium concen- tration. There was no effect of either ViTD or SHADE on the shear force. The L* values were higher at 24 h pm than at 0 and 1 h pm, with no differences among the animals in the ViTD or SHADE groups. Higher a* values were observed among animals in the V8 group than in the V0 group, and higher b* values were observed among animals in the V8 group than in the V2 or V0 groups, which were not different. In conclusion, ViTD and SHADE did not affect animal performance, carcass traits or shear force, whereas animals receiving a lower ViTD dos- age and SHADE exhibited altered myofibrillar fragmentation. ViTD affected the colour param- eters, and changes in the lightness of the beef related to the time pm were found in meat from animals under SHADE.
Vitamin D3 supplementation has been reported to im- prove the tenderisation rate (Foote et al., 2004; Karges et al., 2001; Montgomery et al., 2000; Swanek et al., 1999) and colour (Hansen et al., 2011; Strydom et al., 2007) of meat
by increasing the concentration of calcium in the plasma and muscle as well as the anti-oxidative capacity. However, high doses of vitamin D3 have a negative effect on feed intake (Montgomery et al., 2002; Scanga et al., 2001), average daily gain (Montgomery et al., 2002; Reiling and Johnson, 2003), final body weight, hot carcass weight, and fat thickness (Karges et al., 2001; Reiling and Johnson, 2003). Those effects could eventually be related to vitamin residues in the liver, kidneys, and muscle (Foote et al., 2004; Montgomery et al., 2000, 2002).
In contrast to the results mentioned above, other re- searchers found no benefits of supplementation with vitamin D3 or its metabolites regarding the tenderness (Carnagey et al., 2008; Cho et al., 2006; Pedreira et al., 2003; Reiling and Johnson, 2003; Tipton et al., 2007) or colour (Lawrence et al., 2006; Reiling and Johnson, 2003; Tipton et al., 2007) of the meat. Moreover, results have been reported showing no changes in carcass traits as a result of vitamin D3 supplemen- tation (Montgomery et al., 2002; Swanek et al., 1999).
Although previous studies have investigated the effects of different vitamin dosages, the influence of ultraviolet (UV) radiation from sunlight on the animals has not been consid- ered. This influence may explain inconsistencies in the results regarding vitamin D3 supplementation, especially at lower levels. The 25-hydroxy-vitamin D3 [25(OH)D3] concentra- tions in sheep are affected as the result of the interaction be- tween intravenously administered 25(OH)D3 and sunlight exposure (Hidiroglou, 1987). Excessive sunlight exposure could result in conditioning of the animal to control elevated levels of the active metabolite 1,25-di-hydroxy-vitamin D3 [1,25(OH)2D3]. Negative feedback generated by 1,25(OH)2D3 through renal metabolism has been observed in mice, where any excess of 25(OH)D3 is converted to the inac- tive molecule 24,25-di-hydroxy-vitamin D3 [24,25(OH)2D3] (Omdahl et al., 2002).
This work aimed to verify whether an interaction be- tween levels of vitamin D3 supplementation and sunlight ex- posure conditions could improve the meat tenderisation rate and colour without causing negative effects on animal perfor- mance and carcass traits of B. indicus beef cattle.
- Materials and methods
Forty (412 ± 38 kg) and 41 (411 ± 38 kg) Nellore-type castrated males that originated from a commercial herd with an average age of over 30 months (over four perma- nent incisors) were used to generate the performance and carcass trait data and the meat quality data, respectively. After adaptation to a high-concentrate diet, the animals were divided into three weight groups (light = 376 ± 29 kg, intermediate = 403 ± 16 kg, and heavy= 457 ± 17 kg), which represented the different growth potentials before the beginning of the experimental feedlot period. The animals within each weight group were randomly assigned to the six treatments (three levels of vitamin D3 supplementation × two sunlight exposure conditions). The unshaded and shaded pens occupied different sides of the feedlot; one side of the feedlot had naturally lower in- solation than other side (see Section 2.4).
The animals were allocated to individual pens to allow for measurement of feed intake and to assure vitamin D3 inges- tion. The following experimental treatments were tested: 1) no vitamin D3 supplementation (V0) and no shade (n= 7);
- V0 with shade (50% UV filtration ratio) (n= 7);
- 2 × 106 IU of vitamin D3 for 2 consecutive days pre- slaughter (V2) and no shade (n= 7); 4) V2 with shade
(n= 6 or 7); 5) 2 × 106 IU of vitamin D3 for 8 consecutive days pre-slaughter (V8) and no shade (n= 6); and 6) V8 with shade (n= 7).
- Vitamin D3 supplementation
The vitamin supplement was donated by the DSM Produ- tos Nutricionais Brasil Ltda. (São Paulo, SP, Brazil). Vitamin D3 (4 g= 2 × 106 IU) was mixed with 1 kg of the total diet and provided in a small trough. The vitamin D3 intake was veri- fied through visual observation. After the vitamin D3 intake, the animals were fed the whole ration once a day (morning). The diet was composed of 21% roughage (sugarcane bagasse) and 79% concentrate (60.0% grain corn, 15.2% soybean, 3.8% mineral, and vitamin trace/NC Bov Nutron TMR containing 5636 IU vitamin D3) with the following composition: 80.0% dry matter, 72.1% total digestible nutrients, 13.7% crude pro- tein, 3.0% ether extract, 0.1% calcium, and 0.3% phosphorus.
- Shade and feedlot
Sunshade from Nova Plast Indústria e Comércio Ltda. (Nova Odessa, SP, Brazil) with a UV filtration ratio of 50% was set on the northeast side of the feedlot. This side had nat- urally lower insolation during the experimental period due to orientation of the roof (length) that covers a central corridor between the pens. The sunshade of 50 × 9 m was enough to cover the back and sides of the pens in the feedlot. To block the reflected sunlight that reached the front part of the pens, one area covered by sunshade (50 × 1.5 m) above the feeding trough was used.
The feedlot is composed of two rows of side-by-side pens separated by a central corridor that is 3 m in width, with in- dividual pen areas of 8 m2. In the corridor, the individual troughs are covered by a roof with a height of 4.5 m and a width of 8 m.
- Adaptation to treatments
After a 56-day period of adaptation to a high-concentrate diet, the animals were confined for 45 days with different sunlight exposure conditions. Twenty steers were held in pens without sunshade (the pens on the southwest side), and the other 21 steers were held in shaded pens (the pens on the northeast side). On the 25th day of exposure to differ- ent sunlight exposure conditions, all the animals were condi- tioned to receive part of their diet in a small trough to simulate the vitamin D3 supplementation procedure, before receiving the remaining portion of the diet. On the 37th or 43rd day at the feedlot, the animals began supplementation with vitamin D3 for 8 or 2 days.
- Experimental period and location
The experiment was conducted from 30th November 2007 to 14th January 2008 (summer) in an experimental feedlot located in Andradina (São Paulo state — SP). Andra- dina is located at the coordinates 20°53′45″ South and 51°22′44″ West and has an altitude of 405 m.
- Global and ultraviolet radiation and climate data
The global radiation (GR) data were obtained using a LI200X LI-COR pyranometer (Campbell Scientific Inc., North Logan, UT, USA) located at the Meteorology Centre (São Paulo State University/UNESP) in the city of Ilha Sol- teira, SP. The data are available at http://www.agr.feis. unesp.br/clima/ilha_dez07.htm and http://www.agr.feis. unesp.br/clima/ilha_jan08.htm. The data obtained at Ilha Solteira were used because of the small distance between the cities of Andradina and Ilha Solteira (only 72 km) and the similar climate characteristics (Ilha Solteira is located at the coordinates 20°25′58″ South and 51°20′33″ West and has an altitude of 335 m). The equation used to esti- mate the UV radiation (UVR) in the pens that were naturally exposed to sunlight was “UVR = 0.04155 × GR” (Escobedo et al., 2006). The UVR values estimated for the pens without sunshade were divided in half to estimate the UVR for the pens with sunshade (50% UV radiation filtration). The GR, UVR, and air temperature and humidity (recorded in the pens during the day at 9 am, 12 pm, 3 pm, and 6 pm) con- firm the fair weather observed at the feedlot during the ex- periment (Table 1).
- Animal performance
To study the animal performance, the following variables were recorded: the average feed intake (AFI), the initial (IBW) and final (FBW) body weight, the average daily gain (ADG), and the feed:gain ratio (F:G).
The ingested and refused feed were weighed daily. Addi- tionally, samples of the diet (roughage and concentrate) that was fed and the refused portion were taken at intervals of 20 days to determine the dry matter (DM) content according
Global and ultraviolet (UV) radiation data, and local air temperature and hu- midity during the experimental feedlot period and days of vitamin D3 sup- plementation for the pens without and with sunshade.
to AOAC (1990). Both diet and refusal presented the same DM content, and the feed intake (DM basis) was calculated daily using the equation: (offered diet− refusal) × DM.
The following variables were obtained: AFI=DM intake in 45 days÷ 45 days; ADG=gain (FBW− IBW) in 45 days ÷ 45 days; and F:G= DM intake in 45 days÷ gain in 45 days. The IBW and FBW were obtained by weighing the animals be- fore and after the experimental period, respectively. All the an- imals were submitted to an 18-hour fast prior to each weighing.
- Slaughter and carcass evaluation
Animal slaughter was carried out at a commercial plant under federal inspection from the Ministry of Agriculture. After the slaughter, the carcasses were weighed to obtain the hot carcass weight (HCW). In turn, the hot carcass yield (HCY) was calculated as follows: HCY=(HCW÷ FBW)× 100. The ribeye area (REA) between the 12th and 13th tho- racic vertebrae was recorded using vegetal paper to trace the perimeter of the Longissimus thoracis muscle. The area within the traced perimeter was measured using an LI- 3100 Area Meter (LI-COR Inc., Lincoln, NE, USA). This in- strument is generally used to measure the leaf area of plants. The fat thickness (FT) was measured perpendicular- ly to the surface, between the 12th and 13th thoracic verte- brae, at a lateral position from the animal midline at a point 3/4th of the distance from the medial end of the Longissimus thoracis muscle, as measured using a graded ruler. The REA and FT adjusted to the average HCW were
also included in the analysis.
- Meat samples
After the slaughter, a portion from the Longissimus thora- cis muscle between the 12th and 13th thoracic vertebrae was immediately collected to determine total muscle calcium concentration and meat colour [0 and 1 h post-mortem (pm)]. At 24 h pm, the Longissimus thoracis et lumborum mus-
cle was removed between the 12th thoracic and 5th lumbar vertebrae and cut into nine steaks (2.54 cm in thickness), which were vacuum packaged into 3 packages (3 steaks/ package). The Sulpravac-VC1 packages (Unipac Brasil, SP, Brazil) had medium density (52 g/m2) barrier pouches with 55 micron of thickness and oxygen permeability lower than 10 cm3 m−2 d−1 atm−1 at 0% relative humidity and 23 °C. Following, the steaks were stored at ±2 °C for either 1, 7 or 21 days of ageing. These steaks were used to evaluate the Myofibrillar Fragmentation Index and shear force. One of nine of the steaks that would be aged was randomly selected to record the colour values at 24 h pm.
Legend: NS (no shade)= pens without sunshade in which animals were al- located; WS (with shade)= pens with sunshade (50% ultraviolet filtration ratio) in which animals were allocated; and Day 1 to Day 8 = days of vitamin D3 supplementation. a Data obtained from the equation: UV radia- tion= 0.04155 × global radiation (Escobedo et al., 2006). b Data obtained from the Meteorology Centre of the UNESP (Source: http://www.agr.feis. unesp.br/clima/ilha_dez07.htm and http://www.agr.feis.unesp.br/clima/ ilha_jan08.htm). c Data obtained from the division of global radiation by two, considering the sunshade filtration ratio of 50%. d EP — Experimental period before the vitamin D3 supplementation. e nd — not determined. f Par- tially cloudy sky.
- Carcass pH and temperature
The carcass pH data were recorded at 0, 3, and 24 h pm, and carcass temperature data were recorded at 0, 3, 6, and 24 h pm. Both measures were taken in the Longissimus thora- cis muscle at the left carcass side using a Sentron portable pH and temperature meter (Gig Harbor, WA, USA).
- Meat colour
To determine the beef colour, a CR-400 Minolta Chroma Meter (Minolta Corporation/ISD, Ramsey, NJ, USA) with a 0.8-cm aperture, a D65 light source, and a 2° observer was used to measure the lightness (L*), redness (a*), and yellow- ness (b*) according to the CIELAB system. The readings were taken at the surface of the Longissimus thoracis muscle at 0, 1, and 24 h pm.
- Plasma ionised and total muscle calcium
The blood was collected before and after the period of vi- tamin D3 supplementation. Plasma ionised calcium concen- trations were determined using an i-STAT portable apparatus (Abbot Point of Care Inc., East Windsor, NJ, USA) attached to a CG8+ cartridge. The total muscle calcium con- centration was determined at Analytical Chemistry Laborato- ry by atomic absorption according to Nakamura (1973).
- Warner–Bratzler shear force
The procedure used to analyse the shear force was con- ducted according to the recommendations of the AMSA (1995). Briefly, steaks with 2.54 cm of thickness were cooked on an electric grill (EDANCA®) until reaching internal tem- perature of 40 °C, when they were turned over and cooked to reach internal temperature of 71 °C (monitored by ther- mometers). The steaks were cooled at room temperature and stored overnight at ±2 °C. The next day, six to eight cores with a diameter of 1.25 cm were removed from each steak parallel to the direction of muscle fibres. Then, cores were analysed using the Warner–Bratzler equipment (G-R Manufacturing Co., Manhattan, KS, USA) to measure the force necessary to shear the cores. The results are expressed in kgf.
- Myoﬁbrillar Fragmentation Index (MFI)
Analysis of the MFI was performed with samples that were taken from steaks at 1 and 21 days of ageing and frozen in liquid nitrogen. The homogenisation of 4 g of tissue into MFI buffer using a Waring blender was performed according to the procedure described by Culler et al. (1978). The MFI readings were performed using a spectrophotometer (Cole- man Instruments Division, Oak Brook, IL, USA) at 540 nm.
- Statistical analysis
The experimental design was based on randomised com- plete blocks, where the three different weight groups after adaptation to a high-concentrate diet represented the blocks. An approach was used to analyse two experimental condi- tions (different sunlight exposure conditions) and three dif- ferent levels of vitamin D3 supplementation. To perform this combined analysis, a linear mixed model was used, in which the effect of vitamin D3 supplementation, sunlight ex- posure conditions, and their interactions were considered to be fixed, while the effect of blocks nested within the effect of the sunlight exposure conditions was considered to be ran- dom. For the initial (before vitamin D3 supplementation)
plasma ionised calcium data, the effect of levels of vitamin D3 supplementation and their interaction with sunlight expo- sure conditions were not considered in the model. For the final (after vitamin D3 supplementation) plasma ionised cal- cium data analysis, the initial plasma ionised calcium data were included in the model as a covariate. To account for the correlation that exists among the measurements taken over time in the same experimental unit (animal), the depen- dent variables, such as the carcass pH and temperature, MFI, shear force, and meat colour, were analysed as repeated mea- sures with respect to time. The analyses met the assumptions of the model, where residuals were normally distributed and independent of the data. Each of the six treatments was con- ducted with seven repetitions, except for two treatments (animal performance and carcass traits) or one (meat quality traits) treatment that presented missing data (making a total of 40 or 41 animals) due to problems during the trait mea- surements. The data were analysed using PROC MIXED in a statistical package from SAS (2000) using the Tukey–Kramer test for least squares means contrasts.
- Animal performance and carcass traits
There was no effect (P ≥ 0.23) of the sunlight exposure condition and/or level of vitamin D3 supplementation regard- ing the animal performance or the carcass traits (Table 2). An effect of time pm was detected (P b 0.01, data not shown) for carcass pH and temperature decline as expected. The pH values between 0 h [6.61 (0.054)] and 3 h [6.47 (0.035)] de- creased slightly (P = 0.06), while the pH values between ei- ther 0 h or 3 h and 24 h [5.71 (0.028)] decreased significantly (P b 0.01) to reach a pH value close to the normal average. Carcass temperatures significantly dropped (P b 0.01) across the first 24 h pm [0 h= 27.5 (0.44) °C, 3 h= 19.4 (0.38) °C, 6 h= 13.6 (0.16) °C, and 24 h= 1.2
- Plasma and muscle calcium
The plasma ionised calcium concentration of the animals was not influenced (P ≥ 0.30) by the sunlight exposure condi- tions and/or levels of vitamin D3 supplementation, while the total muscle calcium concentration was altered, showing higher values (P = 0.07) in the animals that were protected by sunshade (Table 3).
- Meat quality
An interaction between the levels of vitamin D3 supple- mentation and sunlight exposure was detected (P = 0.03) for MFI values determined with frozen samples at days 1 and 21 pm (pooled data, Fig. 1), which would reflect frag- mentation extension. Within the animals exposed to sunlight or under sunshade, no differences were observed (P ≥ 0.15) due to the levels of vitamin D3 supplementation. However, higher MFI values were found (P = 0.03) in meat from ani- mals under sunshade compared to those exposed to sunlight within the V2 group.
Least squares means of the animal performance and carcass trait data for dif- ferent levels of vitamin D3 supplementation and sunlight exposure condi- tions during the feedlot period.
Least squares means of the plasma ionised (mg/dL) and total muscle (μg/g wet muscle) calcium concentrations for different levels of vitamin D3 sup- plementation and sunlight exposure conditions.
Sunlight exposure condition
Level of vitamin D3 supplementation V0 V2 V8
Level of vitamin
Sunlight exposure condition Means No shade With shade
Initial body weight (kg)
|No shade||416 (25.2)£||422 (25.2)||411 (25.4)|
|With shade||402 (25.4)||413 (25.5)||406 (25.4)|
|Final body weight (kg)|
|No shade||477 (24.4)||487 (24.4)||489 (24.7)|
|With shade||464 (24.6)||473 (24.8)||466 (24.6)|
Average daily gain (kg/day)
Feed:gain ratio (kg DM/kg BWG)
No shade 7.9 (0.85) 6.7 (0.85) 5.8 (0.92)
With shade 6.3 (0.82) 7.3 (0.89) 7.0 (0.82)
Hot carcass weight (kg)
|No shade||246 (13.4)||249 (13.4)||249 (13.6)|
|With shade||242 (13.3)||243 (13.4)||237 (13.3)|
|Hot carcass yield (%) No shade||
Ribeye area (cm2)
|52.2 (0.44)||51.4 (0.47)||50.8 (0.44)|
|No shade||62.2 (1.79)||65.7 (1.79)||64.0 (1.93)|
|With shade||67.7 (2.40)||65.2 (2.59)||64.8 (2.40)|
|Adjusted ribeye area (cm2)|
|No shade||62.1 (2.45)||64.8 (2.45)||62.8 (2.57)|
|With shade||68.3 (2.54)||65.5 (2.64)||66.5 (2.54)|
|Fat thickness (mm)|
|No shade||2.8 (0.58)||2.6 (0.58)||3.4 (0.62)|
|With shade||3.1 (0.56)||3.3 (0.60)||1.9 (0.56)|
Adjusted fat thickness (mm)
No shade 2.8 (0.51) 2.3 (0.51) 3.0 (0.55)
With shade 3.3 (0.61) 3.4 (0.66) 2.3 (0.61)
Legend: V0 = no supplementation; V2 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 2 consecutive days pre-slaughter; V8 = with vita- min D3 supplementation at a dose of 2 × 106 IU for 8 consecutive days pre- slaughter; no shade= animals allocated into pens without sunshade; with shade= animals allocated into pens with sunshade (50% ultraviolet filtra- tion ratio); DM = dry matter; and BWG= body weight gain. £ Least squares means (standard error).
No effect associated with the levels of vitamin D3 supple- mentation and/or sunlight exposure was found during ageing of the meat (P ≥ 0.15) on the MFI (data not shown) and War- ner–Bratzler shear force values (Table 4). An effect of the du- ration of ageing showing the progress of myofibrillar weakening and a decline in the shear force was observed (P b 0.01).
As expected, the meat colour was affected by the levels of vitamin D3 supplementation (a*, P = 0.06 and b*, P b 0.01) and the time pm (L*, a*, and b*, P b 0.01) (Table 5). The a* values were higher (P = 0.06) for the group that received a higher level of vitamin D3 supplementation than for those that received no vitamin D3 supplementation, while both groups had similar a* values (P ≥ 0.16) as the group that re- ceived the lower level of vitamin D3 supplementation. On the other hand, the b* values were higher (P b 0.01) for the group that received the higher level of vitamin D3
Initial plasma ionised calcium concentration (mg/dL)
Means 4.3 (0.04)£ 4.3 (0.05) –
Final plasma ionised calcium concentration (mg/dL)
|V0||4.4 (0.07)||4.4 (0.07)||4.4 (0.05)|
|V2||4.3 (0.06)||4.4 (0.07)||4.3 (0.05)|
|V8||4.3 (0.07)||4.3 (0.07)||4.3 (0.05)|
|Means||4.3 (0.04)||4.4 (0.04)||–|
Total muscle calcium concentration (μg/g wet muscle)
|V0||54.0 (5.03)||63.2 (10.09)||58.6 (5.64)|
|V2||50.4 (5.03)||67.8 (10.09)||59.1 (5.64)|
|V8||55.7 (5.36)||73.6 (10.09)||64.6 (5.71)|
|Means*||53.4 (3.72)b||68.2 (6.24)a||–|
Legend: no shade= animals allocated into pens without sunshade; with shade= animals allocated into pens with sunshade (50% ultraviolet filtration ratio); V0 = no supplementation; V2 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 2 consecutive days pre- slaughter; V8 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 8 consecutive days pre-slaughter; initial plasma ionised calcium concen- tration= plasma ionised calcium concentration measured before vitamin D3 supplementation; and final plasma ionised calcium concentration = plasma ionised calcium concentration measured after vitamin D3 supplementation.
£Least squares means (standard error). *P = 0.07 for the main effect of sun- light exposure conditions. a,bDifferent lowercase letters between the sun- light exposure conditions indicate a significant difference (P = 0.07).
supplementation than for those that received the lower level of vitamin D3 supplementation or no vitamin D3 supplementa- tion, which were similar (P =0.85).
An interaction (P = 0.03) between sunlight exposure con- ditions and the time pm was verified for the L* values (Fig. 2). In animals exposed to sunlight and animals under sunshade, the L* values were similar (P ≥ 0.79) between 0 and 1 h pm, and they were the highest (P b 0.01) at 24 h pm. No differ- ences were observed (P ≥ 0.16) due to the sunlight exposure conditions with respect to the length of time pm. On the other hand, the sunlight exposure conditions alone affected (P = 0.03, data not shown) the b* values, where meat from animals that were unprotected from sunlight [4.1 (0.17)] had increased (P = 0.03) yellowness compared to meat from animals that were shaded [3.5 (0.17)].
- Animal performance and carcass traits
The lack of a difference between the unshaded and shad- ed animals regarding the animal performance and the carcass traits may be attributed to the beef cattle biological type and intake behaviour. A reduction in animal performance and a decline in the carcass traits among animals exposed to sun- light would be expected due to thermal discomfort (Mitlöhner et al., 2002), however B. indicus cattle are tolerant of a hot climate (Gaughan et al., 1999), and therefore, they may not respond to the benefits of sunshade (Mader et al., 1999). In some cases, it has been observed that animals
Fig. 1. Myofibrillar Fragmentation Index (MFI) from meat frozen in liquid nitrogen at days 1 and 21 post-mortem (pooled data) from animals that received dif- ferent levels of vitamin D3 supplementation and were subjected to different sunlight exposure conditions (interaction — P = 0.03). Legend: □ no shade= animals allocated into pens without sunshade; ■ with shade= animals allocated into pens with sunshade (50% ultraviolet filtration ratio); V0 = no supplementation; V2 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 2 consecutive days pre-slaughter; and V8 = with vitamin D3 supplementation at a dose of 2×106 IU for 8 consecutive days pre-slaughter. aSimilar lowercase letters among the levels of vitamin D3 supplementation within the sunlight exposure condi- tions do not significantly differ (P ≥ 0.15). A,BDifferent uppercase letters between the sunlight exposure conditions within the levels of vitamin D3 supplementa- tion significantly differ (P = 0.03).
exposed to sunlight compensate by ingesting lower amounts of feed during the day but displaying a higher feed intake during the night (Gaughan et al., 2004). The lack of an effect of sunlight exposure conditions on animal performance was previously reported for cattle with B. taurus predominance (Brosh et al., 1998; Mader et al., 1999).
Furthermore, the lack of a difference in the animal perfor- mance and the carcass traits among animals that received vi- tamin D3 supplementation suggests that vitamin D3 given as powder mixed into the diet at a low dosage seems to be not detrimental. Only when vitamin D3 was given via a bolus through a protected gelatinous capsule at the same dosage used in this experiment (Scanga et al., 2001) or fed as a mixed powder at a higher dosage of vitamin D3 (Karges et al., 2001; Montgomery et al., 2002) was there a decrease in feed intake, average daily gain, final body weight, hot carcass weight, and fat thickness.
The levels of vitamin D3 supplementation and sunlight ex- posure conditions did not influence the Longissimus thoracis muscle pH or temperature at the tested time points. These
results corroborate those of another study reporting no changes in the pH values at 3 and 24 h pm and in the temper- ature at 3 h pm after supplementation with several different doses of vitamin D3 (Montgomery et al., 2002). However, the latter study suggested that vitamin D3 supplementation was related to lower carcass temperatures at 24 h pm.
- Plasma and muscle calcium levels
The lack of an effect of vitamin D3 supplementation in dif- ferent sunlight exposure conditions on the plasma ionised calcium concentrations might be explained by the strict con- trol of calcium homeostasis in animals due to its central role in a variety of functions, such as signal transduction and neu- romuscular activities (Littledike and Goff, 1987).
Several other reports also have shown no effect of vitamin D3 and/or its metabolic derivatives on the plasma calcium concentration (Cho et al., 2006; Lawrence et al., 2006; Pedreira et al., 2003; Tipton et al., 2007). In its ionised form, the concentration of calcium in plasma was not changed in
Least squares means of the shear force (kgf) of samples from animals that received different levels of vitamin D3 supplementation and were exposed to different sunlight exposure conditions taken after different durations of post-mortem ageing.
Level of vitamin D3 Sunlight exposure condition
|Day 1||Day 7||Day 21|
|V0||10.0 (0.88)£||10.9 (0.99)||8.7 (0.88)||9.0 (0.99)||7.0 (0.88)||7.6 (0.99)|
|V2||11.6 (0.88)||10.9 (0.99)||10.6 (0.88)||9.3 (0.99)||7.6 (0.88)||7.3 (0.99)|
|10.6 (0.99)||7.6 (0.92)
|9.2 (0.99)||6.0 (0.92)
Legend: NS (no shade)= animals allocated into pens without sunshade; WS (with shade)= animals allocated into pens with sunshade (50% ultraviolet filtration ratio); V0 = no supplementation; V2 = with a vitamin D3 supplementation at a dose of 2 × 106 IU for 2 consecutive days pre-slaughter; and V8 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 8 consecutive days pre-slaughter. £Least squares means (standard error); *P b 0.01 for main effect of post-mortem age- ing periods. a,b,cDifferent lowercase letters among the times of ageing significantly differ (P b 0.01).
Least squares means of the L*, a*, and b* values for samples from animals submitted to different levels of vitamin D3 supplementation determined at different times post-mortem.
25(OH)D3. Those animals were not only deprived of sunlight by the sunshade but also by the feedlot roof (made of asbestos-cement), which had an orientation that would fa- vour the reflectance of a large proportion of the UV radiation
Level of vitamin D3 supplementation Means**
V0 V2 V8
(49.2%, Roma-Jr et al., 2008) from sunlight for most of the day. Low plasma 25(OH)D3 concentrations have been reported in cattle and sheep protected from UV radiation
0 h 30.7 (0.68)£ 29.8 (0.68) 31.2 (0.71) 30.6 (0.40)B
1 h 30.5 (0.68) 29.2 (0.68) 30.6 (0.71) 30.1 (0.40)B
24 h 33.5 (0.68) 32.7 (0.68) 34.5 (0.71) 33.6 (0.40)A
Means 31.6 (0.52) 30.6 (0.52) 32.1 (0.54) –
0 h 14.9 (0.86) 15.5 (0.86) 17.1 (0.91) 15.8 (0.51)B
1 h 14.5 (0.70) 14.6 (0.70) 15.2 (0.72) 14.8 (0.41)B
24 h 17.1 (0.52) 17.5 (0.52) 18.6 (0.54) 17.7 (0.31)A
Means* 15.5 (0.39)b 15.8 (0.39)ab 17.0 (0.41)a –
(Hidiroglou, 1987; Hidiroglou et al., 1979). This scenario is linked to increased parathyroid hormone (PTH) secretion, leading to secondary hyperparathyroidism (Zittermann et al., 2007), which is associated with mobilisation of calcium from the bone matrix. In hyperparathyroidism models, which could result from low plasma 1,25(OH)2D3 concentra- tion, calcium accumulation in the soft tissues has been docu- mented in severe cases (Tamagaki et al., 2006).
- Meat quality
Legend: V0 = no supplementation; V2 = with vitamin D3 supplementation at a dose of 2 × 106 IU for 2 consecutive days pre-slaughter; V8 = with vita- min D3 supplementation at a dose of 2 × 106 IU for 8 consecutive days pre- slaughter; 0 h= 0 hour post-mortem; 1 h= 1 hour post-mortem; 24 h= 24 hours post-mortem; L* = lightness, range from dark to pale; a* = chroma, range from green to red (− a* to + a*); and b* = chroma, range from blue to yellow (− b* to + b*). £Least squares means (standard error). *P = 0.06 and P b 0.01 for main effect of levels of vitamin D3 supple- mentation in a* and b* values, respectively. a,bDifferent lowercase letters among the levels of vitamin D3 supplementation significantly differ for a* (P = 0.06) and b* (P b 0.01) values. **P b 0.01 for main effect of time post- mortem in either L*, a* or b* values. A,BDifferent uppercase letters among the times post-mortem significantly differ (P ≤ 0.02).
lambs (Boleman et al., 2004), although it was increased in cattle (Swanek et al., 1999) supplemented orally with vita- min D3. In both cases, the supplementation doses used were higher than those used in the present study.
The higher total muscle calcium concentration in the shaded animals may be a result of changes in plasma
dosage (V2). The majority of published studies have explained the improvement in myofibrillar fragmentation and/or shear force through the increased free calcium con- centration in the muscle (Montgomery et al., 2002, 2004; Swanek et al., 1999).
On the other hand, the lower immobilised calcium con- centration found in muscle isolated from animals exposed to unlimited sunlight may have been offset by the effects of vitamin D3 supplementation at a high dosage (V8). It has been reported that 1,25(OH)2D3 effectively increased the in- tracellular calcium concentration in chicken skeletal muscle cell culture (Capiati et al., 2000). The increased intracellular calcium concentration would be important for the stimula- tion of calcium-dependent proteases, which have a major im- pact on MFI (Koohmaraie, 1992). In this scenario, a higher muscular concentration of vitamin D3 metabolites could have indirectly enhanced the muscle calcium-dependent protease activities.
Fig. 2. L* values in samples of animals submitted to different sunlight exposure conditions and times post-mortem (interaction — P = 0.03). Legend: □ no sha- de= Animals allocated into pens without sunshade; ■ with shade= animals allocated into pens with sunshade (50% ultraviolet filtration ratio); 0 h= 0 hour post-mortem; 1 h= 1 hour post-mortem; and 24 h= 24 hours post-mortem. a,bDifferent lowercase letters among the times post-mortem within the sunlight ex- posure conditions significantly differ (P b 0.01). ASimilar uppercase letters between the sunlight exposure conditions within the times post-mortem do not signif- icantly differ (P ≥ 0.16).
The ability of vitamin D3 supplementation to weaken the myofibrillar structure and improve tenderness in beef cattle has been observed under conditions that increase the con- centrations of muscle vitamin D3 and its metabolites (Foote et al., 2004; Montgomery et al., 2002), especially 1,25(OH)2D3 (Montgomery et al., 2000). Furthermore, supra- nutritional vitamin D3 supplementation has been associated with increases in the protease mRNAs and activities (Cho et al., 2006; Swanek et al., 1999). In fact, the V8 condition was found to alter the abundance of mRNA encoding one of the calpastatin isoforms, which may reveal a response to higher calpain activity in the hydrolysis of the calpastatin molecule (Rezende, 2011).
It is important to consider the hypothesis that a high 1,25(OH)2D3 concentration in the muscle and regulation of calcium influx in muscle cells may explain the similar myofi- brillar fragmentation levels observed in animals supplemen- ted with the higher level of vitamin D3.
The lack of differences in the MFI and shear force values within each of the ageing times in response to the level of vitamin D3 and sunlight exposure may be a result of limitations in the calpain system. The inhibited calpain proteolysis has been described for the biological type (B. indicus) of the animals used in this study (Shackelford et al., 1991; Whipple et al., 1990). On the other hand, the background tenderness dictated by the collagen content and solubility might have played a relevant role in the lack of improvement in the shear force values in the ani- mals that received vitamin D3 supplementation because the animals were over 30 months of age. The animal age or stage of maturity is also an important factor in shear force measurements due the decrease in collagen solubility that occurs with age (Smith and Judge, 1991; Smith et al., 1988).
Vitamin D3 supplementation has been reported to im- prove tenderness in B. taurus cattle (Karges et al., 2001; Montgomery et al., 2000; Swanek et al., 1999). However, in experiments using B. indicus cattle (Lawrence et al., 2006; Pedreira et al., 2003; Tipton et al., 2007) or old animals such as cull cows (Carnagey et al., 2008; Cho et al., 2006), vi- tamin D3 supplementation has not been shown to improve tenderness.
No post-mortem effect was noted for beef lightness (L* values) among the animals that received different levels of vitamin D3 supplementation or between animals that were exposed to different sunlight conditions, which may be at- tributed to the similar pH values between the groups. The correlation values (r= −0.55 at P b 0.01) between pH and L* values from samples collected at 0 and 24 h pm (pooled data) confirm that beef lightness depends on the pH values, as has been previously observed (Page et al., 2001; Wulf and Wise, 1999). At a higher muscle pH, the meat will be dar- ker in colour because there is less free water to reflect light because proteins bind water more strongly (Ledward et al., 1992; Page et al., 2001). In cattle supplemented with vitamin D3, a lighter colour (Strydom et al., 2007) and minimal light- ness variation (Lawrence et al., 2006; Tipton et al., 2007) have been reported.
The higher values for the chroma parameters (a* and b*)
in samples from animals that received a higher level of vita- min D3 supplementation may be attributed to the possible
anti-oxidative capacity that has been linked to this vitamin (Hansen et al., 2011; Lahucky et al., 2007; Wiegand et al., 2002), which may be related to increased concentrations of antioxidant enzymes (Hamden et al., 2009). However, the lower degree of yellowness found in samples from animals protected from sunlight could be the result of pro-oxidant ac- tivity stimulated by the higher total muscle calcium concen- trations, which were observed in the muscles of those animals. An increasing muscle calcium concentration could increase the number of free radical electrons, providing more catalysts for myoglobin oxidation (Lawrence et al., 2003).
Although the levels of vitamin D3 supplementation affect- ed the chroma parameters, we did not test whether such ef- fects would be perceptible by beef consumers, considering that the differences were small. Within the muscle pH range typical of dark-cutting carcasses (5.6 to 6.8), as oc- curred with 87.5% of the carcasses in our study (pH at 24 h pm: MEAN= 5.72, SD= 0.16, MIN= 5.50, and MAX= 6.22),
the colorimeter values may vary only slightly, whereas mus- cle pH can vary considerably (Wulf and Wise, 1999).
Myofibrillar fragmentation may be influenced by the rela- tionship between levels of vitamin D3 supplementation and sunlight exposure without negatively affecting animal per- formance or carcass traits of beef cattle. Although vitamin D3 supplementation may not be effective for increasing the tenderness of beef from mature B. indicus cattle, it may mod- ify beef colour.
Conﬂict of interest statement
No conflict declared.
This work was funded by “Fundação de Amparo a Pes- quisa do Estado de São Paulo – FAPESP” (Grant # 2006/ 06963-1). The authors acknowledge the beef cattle producer Ms. Luzia Regina Camargo Regazzo as well as Frigo-Ilha Ltda. meat packing plant by helping in the sample collection. We also are grateful to the graduate students Ingrid Monteiro Medina and Flavia Rafaela dos Santos for helping in the labo- ratory analysis.
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