Research Paper EAEF XX(X) : X-X, 2012

Estimation of Serum Level by Color Change of in Japanese Black Cattle

Shuqing HAN*1, Naoshi KONDO*2, Yuichi OGAWA*2, Shoichi MANO*2, Yoshie TAKAO*2, Shinya TANIGAWA*2, Moriyuki FUKUSHIMA*3, Osamu WATANABE*3, Namiko KOHAMA*3, Hyeon Tae KIM *4 , Tateshi FUJIURA*2

Abstract Color change of pupil area was investigated in Red, Green, Blue (RGB) and Hue, Saturation, Intensity (HSI) color models from July to November in 2010 and from May to December in 2011 to estimate the serum vitamin A level of Japanese black cattle during their vitamin A controlled stage. A 2CCD camera was used to acquire the eye images. The results showed lower vitamin A level cattle usually accompanied with higher red component value, lower saturation in their eye images. An estimation model was built based on red and green component ratio. The estimation error is about 10 IU/dL. The reasonable result shows the feasibility to estimate the vitamin A level by color change of pupil area in Japanese black cattle. [Keywords] serum vitamin A, Japanese black cattle, 2CCD camera, tapetum, pupil color, beef quality

1 I Introduction 23 alternative that can estimate vitamin A levels quickly and 24 non-invasively is desirable. 2 To meet the demand in the Japanese market for beef 25 Low vitamin A levels in cattle are known to cause 3 with high marbling scores, Japanese black cattle farmers, 26 retinal degeneration that can lead to increased tapetal 4 generally, control the cattle’s vitamin A level. Studies 27 reflection and loss of pigmentation of the nontapetal 5 have shown that low levels of vitamin A in cattle can 28 fundus (Moore 1941; Vandonkersgoed and Clark 1988; 6 result in a high degree of marbling (Oka et al. 1998; 29 Gelatt 2001). As progresses, the 7 Adachi et al. 1999; Gorocica-Buenfil et al. 2007). 30 disease can occur, where the optic discs 8 Recently, Japanese farmers have begun to active 31 enlarge and become pink and pale. Maggs et al. (2008) 9 manipulate their cattle’s vitamin A level (30-40 IU/dL) 32 showed that mottling of the tapetum and pallor of the 10 during the middle fattening stage: 16 to 24 months (see 33 nontapetum also appear with vitamin A deficiency. 11 Fig.1). Thus, it is important for farmers to monitor the 34 Our laboratory (Takahashi et al. 2011) found a 12 vitamin A level during this middle fattening stage. 35 negative relationship between vitamin A levels and the 13 In addition, maintaining a minimum vitamin A level is 36 reflection of tapetum lucidum, tapetum nigrum and the 14 critical, since a vitamin A deficiency below this level 37 using ultraviolet imaging, suggesting 15 increases disease susceptibility, such as , 38 hyper-reflectivity in cattle at lower vitamin A 16 xerophthalmia, papilledema, or diarrhea (Moore 1941; 39 levels. To our knowledge, this is the first research to 17 O'Donghue 1955; Divers et al. 1986; Mason et al. 2003). 40 estimate vitamin A levels using pupil color change. The 18 The conventional way of measuring vitamin A levels in 41 aim of the current study is to (1) measure changes in 19 cattle is by blood assay. This test is invasive and stressful 42 pupil color, and then (2) estimate the vitamin A levels in 20 to the cattle. Besides that, complicated procedures and 43 Japanese black cattle using the relationship with pupil 21 expensive devices, such as high-performance liquid 44 color during the middle fattening stage. 22 chromatography (HPLC) system are needed. Thus, an

* Partly presented at 127th JSAM Kansai branch meeting in March 2012 *1 JSAM Member, Corresponding author, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. [email protected] *2 JSAM Member, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan *3 Hyogo Prefectural Hokubu Agricultural Institute, Department of Beef Cattle Production, Wadayamacho-Yasui, Asako, Hyogo 669-5254, Japan *4 Dept. of Bio-Industrial Machinery Eng., Gyeongsang National Univ.(Insti. of Agric. & Life Sci.), Jinju 660-701, Korea

2 Engineering in Agriculture, Environment and Food Vol. X, No. X (2012)

120 42 III Devices and Methods 100 43 1. Experimental devices 80 44 A 2CCD multi-spectral camera AD-080CL (JAI) was 60 45 used to acquire both color and Near Infrared (NIR) 40 30 IU/dL 46 images. The camera’s spectral sensitivity is shown in Fig. 20 Vitamin A (IU/dL) A Vitamin 47 2. Light entering the was separated into two light 0 48 beams by a beam splitter; one was focused on a color 8 10 12 14 16 18 20 22 24 26 28 30 32 Age (Months) 49 image sensor and the other was focused on a 1 50 monochrome image sensor. The color image sensor was 2 Fig. 1 Ideal serum vitamin A level in cattle during the 51 a single-chip image sensor with Bayer filters. The 3 fattening stage. The red line indicates the minimum 52 monochrome image sensor was equipped with a filter to 4 desired level (30 IU/dL) of vitamin A in Japanese black 53 capture a NIR image in the wavelength range 5 cattle. For vitamin A, 1 IU is the biological equivalent of 54 750–900nm. The image was 1024 pixels in width and 6 0.3 μg , or of 0.6 μg beta-carotene. (Provided by 55 768 pixels in height. The lens was a TF15D-8 (Focus 7 Hyogo Prefectural Hokubu Agricultural Institute) 56 Length 15 mm, F 2.2, FUJINON) in 2010 and NT63-240 8 57 (Focus Length 12 mm, F 1.8, EDMUND) in 2011. The 9 II Materials 58 camera was combined with two ring-shaped LED lights; 10 This experiment was conducted from July to 59 a MDRL-CW50 (MORITEX) white LED light and a 11 November in 2010 and from May to December in 2011 60 MDRL-CIR31 (MORITEX) NIR LED light which has a 12 with two different groups of cattle at Hyogo Prefectural 61 central wavelength of 850 nm. Two polarizing (PL) 13 Hokubu Agricultural Institute, Japan. 62 filters were installed to reduce the specular reflection 14 In 2010, images of the left eye of 42 Japanese black 63 from the surface of the eye. One of them was installed in 15 cattle were taken at 6 different times. The cattle were 64 front of the LED, the other in front of the camera, 16 made up of 22 Yoshihisa-Yoshinaka-Shigeyasu, 15 65 perpendicular to the former PL filter. A plastic tube was 17 Nobufu-Teruharu, and 5 locally breed cattle. The 66 installed in front of the LED lights to exclude ambient 18 measurements were conducted on July 23rd, September 67 light. The sketch of the device is shown in Fig. 3. Figure 19 15th, October 14th, October 27th, November 10th and 68 4 shows the appearance of the device. 20 November 24th. The cattle’s ages ranged from 15–18 Green Infrared 21 months and at the beginning of the experiment vitamin A 100 Blue Red 22 levels ranged from 17 to 90 IU/dL (mean level 46±20 80

23 IU/dL). （％） 60 24 In 2011, images of the right eye of 42 Japanese black 40 25 cattle were taken once a month. The cattle were made up 26 of 6 breeds (Table 2) and divided into two groups by date 20 27 of arrival. Group A consisted of 24 cattle: “Hiroiwadoi”, Sensitivity 0 28 “Yoshigakidoi” and “Hiromi-Yoshigaki” breeds. They 300 500 700 900 （ ） 29 arrived at the feedlot in December 2010, their ages 69 Wavelength nm 30 ranged from 14–16 months and at the beginning of the 70 Fig. 2 Total spectral sensitivity of camera including 31 experiment their vitamin A levels ranged from 35 to 80 71 prism and sensor. 32 IU/dL (mean level 59±11 IU/dL). Group B consisted of PL filter 33 18 cattle: “Miyagikujyo”, “Kikuyu-Hiromasa Ring –shaped NIR LED light (850 nm) Tube 34 -Miyagikujyo” and “Kikuyu- Hiromasa” breeds. They 35 arrived at the feedlot in February 2011. Their ages 36 ranged from 12–14 months, and at the beginning of the 2CCD Camera

37 experiment their vitamin A levels ranged from 35 to 80 Cattle eye Cattle 38 IU/dL. From the beginning of April 2011, the cattle were Lens（12mm） 39 subjected to a vitamin A free-diet. All the cattle used in Sponge 40 this study were clinically healthy during the 72 Ring -shaped white LED light 41 experimental period. 73 Fig. 3 Sketch of experimental device.

HAN, KONDO, OGAWA, TAKAO, TANIGAWA, FUKUSHIMA, WATANABE, KOHAMA, KIM, 3 FUJIURA: Estimation of Serum Vitamin A Level by Color Change of Pupil in Japanese Black Cattle

27 information. The yellow color in figure 6 shows the 28 region of interest (pupil area). The region of interest was 29 determined by the hue value of the color image and the 30 brightness of the NIR image. As shown in figure 5, very 31 little NIR light was reflected from the tapetum, hence the 32 NIR images were not used in any subsequent analysis. 1 2 Fig. 4 Appearance of experimental device.

33 34 Fig. 6 Original image (Left), Region of Interest shown 3 35 in yellow color (Right). 4 Fig. 5 Color and NIR images of cattle’s right eye. 36 5 37 A comparison of the color change in RGB color model 6 2. Method 38 and HSI color model based on Gonzalez et al. (1992) 7 Color calibration was performed prior to each 39 was conducted. Red (r), green (g), and blue (b) 8 experiment to ensure high quality images and minimize 40 component ratios were calculated by the formulas below: 9 operation error. System setting and color calibration was 41 r = R/(R+G+B) (1) 10 carried out according to the procedure shown in Table 1. 42 g = G/ (R+G+B) (2) 11 43 b = B/ (R+G+B) (3) 12 Table 1 Machine vision system setting procedure. 44 where R, G and B represent red, green and blue, 45 respectively. They have been normalized and are in the NO. Setting 46 range [0, 1]. The HSI color space is claimed to be the 1 Set the brightness at the end of tube to 1700 lx 47 closest approximation to a human interpretation of colors. 2 Set the focus to 8 mm from the end of tube 48 There are many different models for HSI color space. 3 Set F value to 2.2 in 2010 and 1.8 in 2011 49 Here, we use the triangular pyramid model. The 4 Set shutter speed to 1/30 s 50 calculation method is shown below.

5 Set master gain to 336 51 6 Check white balance and calibrate camera by

adjusting red and blue gain

7 Take images of Color Checker (color calibration

board)

13

14 After calibration, the cattle’s eyes were covered for

15 two minutes to adapt to a dark environment. This black 16 cloth cover was kept on until the eye images were 17 captured. Then, we adjusted the aim of the camera to the 52 To make “Saturation” and “Intensity” easy to interpret, 18 cattle’s eye using NIR-LEDs lighting. After a focused 53 they are multiplied by 100, and 255, respectively. So 19 image of the pupil was obtained on the NIR video 54 they are in the range of [0,100] and [0,255], respectively. 20 monitor, the white LEDs were turned on and the eye 55 IV Results 21 images were captured simultaneously. Figure 5 shows 56 1. Relationship between individual color factors 22 sample images. Blood assays were conducted one to two 57 and serum vitamin A in 2010 23 days before image acquisition. Vitamin A levels in the 58 In 2010, the vitamin A level in all the cattle declined 24 blood were determined by HPLC. 59 and then stayed at a low level around 20–30 IU/dL. By 25 The images were processed by software developed in 60 the end of the experiment, the cattle’s vitamin A levels 26 Microsoft Visual Studio 2008 to obtain the color 61 ranged from 7 to 35 IU/dL (mean level 19±7 IU/dL).

4 Engineering in Agriculture, Environment and Food Vol. X, No. X (2012)

1 Figure 7 shows changes in vitamin A (mean ± std) and July 23rd November 24th 2 r (mean ± std) over time. This indicates that when 0.27 3 vitamin A drops, r would increase. 0.26 0.27 80 0.25 70 0.26 0.24 60 r 0.25 0.23 50 0.24 0.22 r 40 0.23 0.21 30 0.20 0.22 20 0 20 40 60 80 100

0.21 10 Vitamin A(IU/dL) Vitamin 0.20 0 Vitamin A (IU/dL) 34 35 Fig. 8 Relationship between red component ratio and r Vitamin A 4 36 vitamin A level in July 23rd and November 24th 2010. 5 Fig. 7 Changes in vitamin A and red component ratio July 23rd November 24th 6 from July to November 2010. Error bars indicate the 40 7 standard error of mean (n=42). 35 8 30

9 In 2010, only average vitamin A levels on July 23rd S 25 10 (mean level 46±20 IU/dL) were significantly above 30 20 11 IU/dL. In subsequent months, levels were around or 15 12 below 30 IU/dL. A comparison between data on July 0 20 40 60 80 100 13 23rd and November 24th was conducted. This Vitamin A (IU/dL) 14 represents the highest and lowest levels of vitamin A, 37 15 respectively. The results are shown in Fig. 8. The r on 38 Fig. 9 Relationship between saturation and vitamin A 16 July 23rd were significant lower than that on November 39 level in July 23rd and November 24th 2010. 17 24th (p<0.001). This indicates there is a negative 40 18 correlation between r and vitamin A. Saturation was 41 2. Relationship between individual color factors 19 also investigated in the same way. The results show that 42 and serum vitamin A in 2011 20 vitamin A deficient in cattle is usually accompanied by 43 Vitamin A levels in all the cattle gradually declined 21 a lower saturation value, as shown in Fig. 9. The 44 during the experimental period in 2011. By the end of the 22 significant color change in the pupil area indicates that 45 experiment, the experiment cattle’s vitamin A levels 23 changes in the tapetum and induced by low 46 ranged from 11 to 41 IU/dL (mean level 28±7 IU/dL). 24 vitamin A levels can be detected by measuring the 47 The relationship between vitamin A level and r, g, b, 25 reflected light from the back of the pupil. 48 hue, saturation and intensity were investigated. Figure 10 26 To confirm these findings, a similar experiment was 49 shows the correlation between r and vitamin A level 27 conducted again in 2011 with another group of cattle. 50 from May (12–16 months old) to December (19–23 28 This time the experimental period was extended from the 51 months old) 2011. The correlation is weak. Similar to the 29 5 months to 8 months in order to cover a wider range of 52 results in 2010, lower vitamin A levels tend to be 30 vitamin A levels. In addition, a new lens with a smaller F 53 associated with a higher red component. As the vitamin 31 value (1.8) was used in 2011 to acquire brighter eye 54 A level dropped, the red color component value 32 images, and thus make it easier to detect color changes. 55 increased. The changes in vitamin A level (mean ± std) 33 56 and r (mean ± std ) over time are shown in Fig. 11. This 57 data also show that as vitamin A levels decline, r rises.

HAN, KONDO, OGAWA, TAKAO, TANIGAWA, FUKUSHIMA, WATANABE, KOHAMA, KIM, 5 FUJIURA: Estimation of Serum Vitamin A Level by Color Change of Pupil in Japanese Black Cattle

0.34 y = -0.0003x + 0.2951 32 levels, especially in October, November and December 33 (p<0.01), as shown in Table 3. It is possible that Group B 0.32 R² = 0.1816 34 had more vitamin A storage in their body, so the time 0.3 r 35 taken to deplete vitamin A stores took longer. Which in 0.28 36 turn means the detection of color changes will take

0.26 37 longer. 38 Table 2 Correlation coefficients between color factors 0.24 0 20 40 60 80 100 39 and vitamin A levels. Vitamin A (IU/dL) 1 Breeds Number r g b H S I 2 Fig. 10 Relationship between red component ratio and of cattle 3 vitamin A level. Hiroiwadoi 8 0.52- 0.76+ 0.23+ 0.86- 0.56+ 0.68- 0.30 80 - + + - + - 70 A Yoshigakidoi 4 0.75 0.50 0.65 0.82 0.75 0.58 0.29 60 Hiromi-Yoshigaki 12 0.68- 0.29- 0.56+ 0.70- 0.68+ 0.57-

0.28 50 - + + - + -

r Group A 24 0.60 0.51 0.42 0.77 0.62 0.55 40 - - + - + - 0.27 30 Miyagikujyo 4 0.27 0.09 0.29 0.05 0.28 0.65 20 - + + - + - 0.26 B Kikuyu-Hiromasa 5 0.39 0.08 0.30 0.21 0.39 0.24 10 Vitamin A(IU/dL) Vitamin -Miyagikujyo 0.25 0 - + + - + - MAY JUN JUL AUG SEP OCT NOV DEC Kikuyu-Hiromasa 9 0.17 0.44 0.01 0.45 0.17 0.65

- + + - + - 4 r Vitamin A Group B 18 0.21 0.28 0.09 0.33 0.21 0.55 5 Fig. 11 Changes in vitamin A and red component ratio Overall 42 0.42- 0.38+ 0.27+ 0.55- 0.42+ 0.55- 6 from May to December 2011. Error bars indicate the 40 7 standard error of mean. 41 Note: “+” indicates positive correlation; “-” indicates negative 8 42 correlation. 9 According to past veterinary experience, at the 43 Table 3 Serum vitamin A levels from August to 10 beginning of the fattening stage serum vitamin A levels 11 of cattle will be around 80 IU/dl. After 3 months of a 44 December. 12 vitamin A-free diet, vitamin A levels will be around the Breeds NO. Aug Sep Oct Nov Dec 13 critical 30 IU/dl value. The symptoms of vitamin A Hiroiwadoi 8 44±5 45±6 25±3 26±3 26±5 14 deficiency may not show for another 3 months. Based on A Yoshigakidoi 4 44±4 45±6 28±3 21±2 25±4 15 this information, we choose the data in May, August and Hiromi-Yoshigaki 12 39±8 39±5 26±5 24±4 24±4 16 December; corresponding to the beginning, middle and Group A 24 42±9 42±7 26±5 24±4 25±6 17 end of middle fattening stage, for correlation analysis. 18 The correlation between color factors (H, S, I, r, g, b) Miyagikujyo 4 57±6 47±5 39±5 37±4 31±3 19 and vitamin A level in the selected three months were B Kikuyu-Hiromasa 5 58±5 54±8 39±6 39±7 36±4 20 investigated. Table 2 shows the correlation coefficients -Miyagikujyo 21 between color factors and vitamin A levels. In Group A, Kikuyu-Hiromasa 9 62±10 50±7 33±7 35±8 28±6 22 the correlation coefficients of r, S, H were higher than Group B 18 60±10 51±9 36±9 37±8 31±7 23 0.6, indicating a strong correlation. Generally, cattle with Overall 42 49±13 46±9 30±9 30±9 28±7 24 lower vitamin A levels had higher r, H, I and lower b, S, 45 25 as Table 2 shows. This result for r and S was consistent 46 Unit: IU/dL. 26 with the result obtained in 2010. This higher correlation 47 27 was obtained using the HSI color model. 48 3. Result of Multiple Linear Regression (MLR) 28 Group B showed a weak correlation between color 49 Analysis 29 factors and vitamin A levels, where all the correlation 50 To estimate vitamin A levels, a MLR model of vitamin 30 coefficients were lower than 0.5. Compared to Group A, 51 A levels and color factors was developed in Excel 2007. 31 cattle in Group B had significantly higher vitamin A 52 To build the calibration models, the data at three sampled

6 Engineering in Agriculture, Environment and Food Vol. X, No. X (2012)

1 dates (May, August and December) for sixteen Group A 31 measure of the degree to which pure color is diluted by 2 cattle were used, and the data at three sampled dates 32 white light (Gonzalez et al. 1992), may indicate more 3 (May, August and December) for 8 Group A cattle were 33 white LED light is reflected by the tapetum when there is 4 used for validation. The results are shown in Table 4. 34 vitamin A deficiency. As the tapetum is blue in color, a 5 Table 4 Multiple Linear Regression Models. 35 low blue component value for cattle with a low vitamin 36 A level may indicate the mottled appearance of the 2 Color Factors MLR Model R 37 non-tapetal area because of retina degeneration (Maggs r,g 푦 = 915푥푟 + 2372푥𝑔 465 0.67* 38 et al. 2008). However, this result is based on the 6 39 subjective notion that the main factor impacting color 7 Note: * p < 0.05, where xr, xg, is the value of r, g respectively; y is 40 changes in the eyes of Japanese black cattle is associated 8 the estimated serum vitamin A level. 41 with vitamin A levels. Further research, with a vitamin A 9 42 supplement group, is need to confirm pupil color 10 The “r, g” model was used to estimate the vitamin A 43 changes are caused by vitamin A levels, and not other 11 values of the other 8 cattle. The performance of the “r, g” 44 factors, such as age. 12 model was evaluated in terms of root mean square error 45 The RMSEP (about 10 IU/dL) may be affected by the 13 of calibration (RMSEC) and root mean square error of 46 individual differences between cattle. To decrease the 14 prediction (RMSEP), and the coefficient of 47 effect of individual differences on the estimation error, it 15 determination (R2). The results are shown in Table 5. 48 may be effective to conduct a blood assay of vitamin A 16 Scatter plots of the “r, g” models for calibration and 49 levels at the beginning of vitamin A control stage. Thus, 17 validation are shown in Fig. 12. The data are evenly 50 the estimation error could be reduced based by taking 18 distributed around the regression line with a 45º slope, 51 into account color changes and initial vitamin A levels. 19 which indicates estimated and measured values are very 52 According to the Japan Livestock Technology 20 close to each other. 53 Association, three cattle blood assays are needed to 21 54 identify vitamin A deficiency during the middle fattening 22 Table 5 Result of “r, g” estimation model. 55 stage. With the suggested sampling regime, blood assays Model RMSEC R2(Calibration) RMSEP R2(Validation) 56 could be reduced to 1. Issi reported that the loss of (IU/dL) (IU/dL) 57 pupillary light reflexes is also a clinical sign of vitamin A r, g 9.3 0.67 10.3 0.62 58 deficiency (Issi and Gül 2010). To make the estimation 59 procedure more robust and accurate, other factors such 80 60 as pupillary light reflex and surface reflection, which 61 may be affected by low vitamin A levels, should also be 62 investigated in the future. 60 63 VI Conclusions

40 64 A negative correlation between the red component 65 ratio of cattle pupils and vitamin A levels, a positive 66 correlation between the saturation value of cattle pupils 20 67 and vitamin A levels were found in 2010, and confirmed

Estimated vitamin A (IU/dL) A vitamin Estimated 68 in 2011. For 24 cattle, with a wider range of vitamin A 0 69 levels than that in 2010, a multiple linear regression 0 20 40 60 80 70 analysis was conducted, and a model y= Measured vitamin A (IU/dL) 71 -915xr+2372xb-465 based on the red and green Validation Calibration 23 72 component ratio was selected. The coefficient of 24 Fig. 12 Measured and estimated vitamin A by “r, g” 73 determination for calibration and validation was 0.67 and 25 model. 74 0.62, respectively. RMSEC was 9.3 (IU/dL) and RMSEP 26 75 was 10.3 (IU/dL). This model may not be applicable to 76 other cattle breeds, but the results show the feasibility of 27 V Discussion 77 estimating vitamin A levels using the color signatures of 28 The result shows that cattle with a lower vitamin A 78 Japanese black cattle during the middle fattening stage, 29 level usually have a lower pupil saturation and lower 79 when their vitamin A levels are depleted. This 30 blue component value. The decreased saturation, a

HAN, KONDO, OGAWA, TAKAO, TANIGAWA, FUKUSHIMA, WATANABE, KOHAMA, KIM, 7 FUJIURA: Estimation of Serum Vitamin A Level by Color Change of Pupil in Japanese Black Cattle

1 information derived from pupil color changes will be a 48 relationship to vitamin A deficiency. Canadian Journal of 2 valuable aid to farm management. 49 Comparative Medicine 19(2): 61-64. 3 50 Oka, A., Y. Maruo, T. Miki, T. Yamasaki, T. Saito. 1998. 51 Influence of vitamin A on the quality of beef from the 4 Acknowledgement 52 Tajima strain of Japanese black cattle. Meat Science 5 This study was supported by Grants-in-Aid for 53 48(1-2): 159-167. 6 Scientific Research (23380153). We gratefully 54 Takahashi, N., N. Kondo, O. Watanabe, M. Fukushima, F. Iwaki, 7 acknowledge the financial support of Ministry of 55 N. Q. Tuan, K. Yoshida, T. Shiigi, Y. Ogawa, K. Fukuzono, 8 Education, Culture, Sports, Science and Technology. We 56 M. Nakano. 2011. Effects of serum vitamin A level on 9 also want to thank the staff of Hyogo Prefectural Hokubu 57 tapetum in Japanese black cattle eye. Engineering in 10 Agricultural Institute for their help and support with the 58 Agriculture, Environment and Food 4(4): 126-130. 11 cattle. We really appreciate Mark De Kleine for his 59 Vandonkersgoed, J. and E. G. Clark 1988. Blindness caused by 12 revision of this manuscript, Tomoo Shiigi and other 60 hypovitaminosis-A in feedlot cattle. Canadian Veterinary 13 laboratory members for their help during the experiment. 61 Journal 29(11): 925-927. 14 We thank Professor Garry Piller for his proof reading of 62 (Received : X. August. 2012) 15 this paper. 16 17 References

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