Livestock Research for Rural Development 27 (5) 2015 Guide for preparation of papers LRRD Newsletter

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Supranutritional vitamin D3 does not affect blood and muscle calcium level, and pre-rigor meat tenderization of spent chicken

A Abd-Hamid1, A Q Sazili1, 2, K Nakyinsige2, 5, A R Alimon1, M Z A Ab-Kadir3 and S A Karsani4

1 Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
2 Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
3 Department of Electric and Electronic, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
4 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
5 Department of Food Science and Nutrition, Islamic University in Uganda, P.O. Box 2555, Mbale, Uganda.


There are numerous pre and post slaughter techniques that could influence meat tenderness. Increased levels of intracellular calcium by inclusion of vitamin D3 to increase absorption of calcium from the bone and intestine are among the pre slaughter interventions for improving meat tenderness. At present, dietary supplementation of different level of vitamin D3 on spent chicken associated with calcium concentration and meat tenderization are yet to be studied. An experiment was therefore conducted to determine the effect of vitamin D3 supplementation on Ca²+ concentration (in muscle and blood) and meat tenderness of spent chicken. One hundred 80 weeks old spent layer were used as experimental birds. The birds were fed with commercial layer feed for one week during adaptation period. Prior to commencement of the experimental diets, blood for the determination of serum calcium analysis was withdrawn. Different supplementation levels (0, 25 x 10, 50 x 10, and 1 x 105 I.U.) of vitamin D3 were mixed with commercial finisher layer diets. After one week of feeding with experimental diets, the birds were slaughtered and blood samples for serum Ca²+ analysis were collected. The breast muscles were dissected out and assigned for muscle Ca²+ determination and shear force analysis.

Supplementation with 0, 25 x10, 50 x 10, 1 x 105 IU of vitamin D3 decreased (p<0.05) serum Ca²+ concentration. Supplementation with vitamin D3 does not give any significance increased in muscle calcium. In addition, shear force values of the breast muscles increased with vitamin D3 supplementation. These results indicated that vitamin D3 supplementation for 7 days does not elevate serum Ca²+, muscle Ca²+ concentrations, and meat tenderness of spent chickens.

Keywords: calpain, layer diet, myofibrillar proteolysis, poultry


The poultry industry is the most important sector in the Malaysia livestock industries. This is shown by the increasing per capita consumption in 2013 compared to 2004 (46.6 and 33.6 kg respectively). The retail price of spent chickens in 2012 was as low as RM 6.85/kg whereas that of processed chicken was as high as RM 7.40/kg (DVS 2013). This shows that the economic value of spent chicken is considerably low. The meat and offal derived from spent chicken are commonly subjected to further processing and rendering for the pet food industry rather than being used for human consumption (Navid et al 2011; Navid et al 2010).

It is well established that toughness of spent chicken meat is primarily due to the increased cross linking of the connective tissue in older birds (Archile-Contreras et al 2011; Wattanachant et al 2004; Bailey and Light 1989). Several studies have been conducted on the tenderization of spent chicken meat through pre and post slaughter interventions (Abdalla et al 2013; Navid et al 2011; Navid et al 2010; Bhaskar et al 2006; Mendiretta et al 2002; Naveena and Mendiretta 2001). Vitamin D3 is a fat soluble vitamin that is usually metabolized in the liver. Supplemented vitamin D3 will be absorbed through the small intestine and be transported in the blood to the liver, where the metabolite will undergo conversion into 25-hydroxycholecalciferol. The 25-hydroxycholecalciferol will then be further converted into 1, 25-dihydroxycholecalciferol, which is an active form of the metabolite that is synthesized in the kidney. Previously, pre slaughter supplementation of vitamin D3 has been implicated in increased muscle calcium concentration and the resultant enhancement of the calpain proteolytic system which has been linked to improved meat tenderness in cattle (Montgomery et al 2002), sheep (Boleman et al 2004) and pigs (Enright et al 1998). However, previous reports on the effects of vitamin D3 supplementation on spent chicken tenderness were only limited to physico-chemical properties (Navid et al 2010). Therefore, the current study was carried out to determine the effects of different levels of vitamin D3 supplementation on Ca+ concentration in blood and muscle as well as the pre-rigor meat tenderization of spent chicken.

Materials and methods

Experimental procedure and dietary treatments

One hundred spent chicken at 80 weeks old and of uniform live weights (2.00.2kg) were randomly divided into 4 dietary treatment groups, namely C0 (commercial finisher layer diet; without addition of vitamin D3), C25000 (commercial finisher layer diet + 25 x 10 I.U. vitamin D3), C50000 (commercial finisher layer diet + 50 x 10 I.U. vitamin D3) and C100000 (commercial finisher layer diet + 1 x 105 I.U. vitamin D3) (Table 1). The finisher layer feed was obtained from a commercial feed miller while vitamin D3 was purchased as Bio D Feed Premix (Gladron Malaysia). Twenty five birds were allotted to each treatment, with 5 replicates per treatment (each consisted of 5 birds). After 1 week of the adjustment period during which the birds were fed the control diet (commercial finisher layer diet), the birds were fed ad libitum for 1 week with the experimental diets. Blood samples were collected from each bird prior to the commencement of the feeding experiment. Feed intake and body weight gain were monitored throughout the feeding trial. The birds were weighed individually upon arrival, prior commencing feeding experimental feed and also prior to slaughter. The feed intakes were measured by weighing the feed before and after feeding experimental diets.

Table 1. Chemical composition of dietary treatments.
Chemical composition Dietary treatments
C0 C25000 C50000 C100000
Crude protein (%) 16 16 16 16
Crude fibre (%) 6 6 6 6
Crude fat (%) 3 3 3 3
Ash (%) 14 14 14 14
Moisture (%) 13 13 13 13
Calcium (%) 3 3 3 3
Vitamin D3 (I. U./ kg DM feed) 0 25 x 10 50 x 10 1 x 105
C0, spent chickens not supplemented with vitamin D3; C25000, spent chickens supplemented with 25 x 10 IU of vitamin D3 per kg DM of feed; C50000, spent chickens supplemented with 50 x 10 IU of vitamin D3 per kg DM of feed; C100000, spent chickens supplemented with 1 x 105 IU of vitamin D3 per kg DM of feed.
Serum sampling

Blood samples from each individual animal were collected prior commencing feeding experiment diet and again during slaughtering. Samples were collected aseptically through wing venipuncture, using 23- gauge needles into a 10 ml Vacutainer (BD Franklin Lakes NJ USA) serum tubes which were later kept slanting for 1 h, followed by centrifugation at 3,000 g for 10 min. The resulted serum was frozen at -20 C until subsequent serum calcium determination.

Slaughter and muscle sampling

Upon completion of the feeding trial, the birds were transported to a research abattoir located in UPM for slaughtering, processing and sampling. Blood samples were collected during slaughtering and stored at -20 C until subsequent calcium concentration determination. The birds were slaughtered according to halal procedure in compliance with the MS1500:2009 (Department of Standards Malaysia). Samples of breast muscles (Pectoralis major m.) were dissected out and stored in a freezer (-80 C) for shear force (objective method to determine meat tenderness) and calcium concentration determinations.

Shear force analysis

Shear force assessment was carried out according to the Volodkevitch shear force determination procedure by Nakyinsige et al (2014). Frozen samples of chicken breast meat were thawed overnight at 4 C. The samples were packed in water impermeable polyethylene bags and cooked in 80 C water bath for 20 min until the core temperature of the steak reached 80 C. After an overnight cooling at 4 C, the samples were cut into cubes with dimension of 1 cm (height), 1 cm (width), and 2 cm (length). The samples were sheared in the centre and perpendicular to the longitudinal direction of the fibres using Volodkevitch bite jaw attached to a texture analyzer machine (TA-XT2i, Stable Micro System, U. K.). The force required to shear the samples was determined in kilogram.

Serum calcium determination

Total free calcium were determined by following Sazili (2004) method. Frozen serum samples were thawed in a water bath (40 C) for a few sec prior to mixing using a vortex. The serum was diluted 25 fold using ultrapure water (Direct Q3UV-R, Merck Millipore, Germany) and analyzed for calcium concentration using Atomic Absorption Spectrometer (AAS) (Perkin Elmer, Pin Aacle 900T, USA).

Muscle calcium determination

Muscle calcium determination was carried out following the procedure of Sazili (2004). Frozen muscle tissues were manually pulverized in liquid nitrogen (Malaysia Oxygen Berhad) by using mortar and pestle. One gram of crushed tissue was weighed and were ashed with lids on in a furnace at 600 C for 12 h. After cooling, the samples were then subjected to acid digestion in 1 ml of 6 M HCl. The samples were further diluted for 50 fold with ultrapure water (Direct Q3UV-R, Merck Millipore, Germany) to ensure that the calcium content fits within the calibration range.

Statistical analysis

The data were subjected to one way ANOVA by the GLM procedure of SAS (SAS version 9.3, SAS Institute Inc.). The differences between means were determined by using Duncan multiple-range test at a significance level of p<0.05.

Results and Discussion

The results for the effect of different levels of vitamin D3 supplementation on calcium concentrations in serum and muscle, and meat tenderness are as presented in Table 2. Blood analysis showed that the C0 (commercial finisher layer diet; without addition of vitamin D3) had the highest serum calcium concentration (23.9 mg/dL) at p<0.05, and as the level of vitamin D3 inclusion increased C25000 (commercial finisher layer diet + 25 x 10 I.U. vitamin D3 C50000 (commercial finisher layer diet + 50 x 10 I.U. vitamin D3) and C100000 (commercial finisher layer diet + 1 x 105 I.U. vitamin D3), the amount of calcium detected in the blood decreased 22.6, 22.3, 21.9 mg/dL, respectively (Table 1). The present results of serum Ca²+ contradict the earlier findings reported by Tipton et al (2007), Scanga et al (2001), and Swanek et al (1999) that serum calcium significantly increased in beef cattle fed supranutritional levels of vitamin D3. Tipton et al (2007) reported that serum calcium concentration was increased in cattle after supplement removal for 7 days, but not immediately following supplementation. Total extractable calcium in muscle tissues are displayed in Table 2. Additionally, different levels of vitamin D3 supplementation did not show any influence on muscle calcium (p>0.05). This contradicts with the results observed by Swanek et al (1999) but follows as reported by Tipton et al (2007). Swanek et al (1999) observed M. longissimus lumborum samples from vitamin D3 supplemented steers to have higher water extractable calcium levels than controls; however Tipton et al (2007) observed for vitamin D3 cattle were somewhat lower in water extractable calcium levels than control cattle. Shear force assessment showed that the control group (C0 -without addition of vitamin D3) displayed the lowest value (p<0.05), whereas increasing the vitamin D3 level C25000 (commercial finisher layer diet + 25 x 10 I.U. vitamin D3), C50000 (commercial finisher layer diet + 50 x 10 I.U. vitamin D3) and C100000 (commercial finisher layer diet + 1 x 105 I.U. vitamin D3) increased the shear force of the breast muscle (1.17 kg, 1.15 kg, and 1.30 kg, respectively) (Table 2).

In the present study, the results of shear force values contradicted with those reported earlier in spent chicken (Navid et al 2010) and steers (Molema M S 2007; Foote et al 2004; Pedreira et al 2003; Montgomery et al 2000; and Swanek et al 1999). However, the results were consistent with the results of Mabelebele et al (2012) which is vitamin D3 supplementation had no effect on tenderness, juiciness, and flavor of unaged Venda cock meat. Similarly, Ma et al (2009) reported that supplementation of vitamin D3 had no significant effect on growth performance and also muscle tenderness of yellow chicken. In addition, Rider et al (2004) and Scanga et al (2001) also reported similar findings as in the present study whereby supplementation of vitamin D3 had no effect on Warner Bratzler shear force values of beef steaks. Scanga et al (2001) reported that oral supplementation with vitamin D3 increased serum Ca²+ concentration but did not improve cooked longissimus tenderness. In this study, the dietary supplementation vitamin D3 did not improve meat tenderness. This was evident by the shear force values which remained unaffected by the treatment. In addition, Ca²+ level in the blood was also found to be unaffected by the supra vitamin D3 supplementation. The unperturbed that the supranutritional vitamin D3 supplementation employed in this study has not effected in any enhancement of the calpain proteolytic system which has been linked to improved post mortem meat tenderization. In addition, older birds tend to have more cross linking of the connective tissue and even after heat denaturation the cross-linked collagen may still be insoluble and this may subsequently results in loss of moisture and meat toughening (Archile-Contreras et al 2011; Wattanachant et al 2004; Bailey and Light 1989). The contradiction of the effects of vitamin D3 in improving meat tenderness in our findings can most probably due to the negative feedback mechanism as it did not allow a drastic resorption in bone and also absorption in the intestine to increase Ca²+ level in the blood. It could because of duration of 7 days feeding treatment would have allowed homeostatic mechanism feedback takes place.

Table 2. Effect of different levels of Vitamin D3 supplementation on blood and muscle calcium concentrations and meat tenderness
Parameters Dietary treatments SEM P value
C0 C25000 C50000 C100000
Blood calcium (mg/dL) 23.9a 22.6ab 22.3b 21.9b 0.26 <0.05
Muscle calcium (g/g) 319a 298a 272a 249a 55.2 NS
Shear force (kg) 1.02b 1.17ab 1.15ab 1.30a 0.0771 NS
a b c Means SE within a row with different superscripts (s) differ significantly at p<0.05


The present results show that 7 day of vitamin D3 supplementation in spent chicken does not increase serum and muscle calcium levels and these may help in explaining the unaffected meat tenderness associated with calpain mediated myofibrillar proteolysis. Therefore, future study on shorter period of supranutritional vitamin D3 supplementation to spent chicken is recommended.


Special thanks are extended to colleagues of Meat Science Laboratory, Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia for the assistance throughout this project.


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Received 20 December 2014; Accepted 10 April 2015; Published 1 May 2015

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