Statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic measurement at 30-month-old of Hanwoo steers<sup>†</sup>

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(1)Journal of the Korean Data & Information Science Society 2018, 29(6), 1721–1729. http://dx.doi.org/10.7465/jkdi.2018.29.6.1721 한국데이터정보과학회지. Statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic † measurement at 30-month-old of Hanwoo steers Jae Jung Ha1· Yun Sook Jung2· Byung Ki Kim3 · Dae Jin Jung4 · Dong Yep Oh5 · Dae Hyun Kim6 · Jea Young Lee7 · Myoung Ok Kim8 · Oh Kyu Kwon9 · Sung Hyun Kim10· Jun Koo Yi11. 123456. Gyeongsangbuk-Do Livestock Research Institute Department of Preventive Dentistry, Kyungpook National University 7 Department of Statistics, Yeungnam University 8 Department of Animal Biotechnology, Kyungpook National University 9 Department of Ecology and Environmental Science, Kyungpook National University 10 China-US(Henan) Hormel Cancer Institute 11 Gyeongsangbuk-Do Livestock Research Institute 2. Received 13 September 2018, revised 1 November 2018, accepted 13 November 2018. Abstract This study was conducted to produce a statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic measurement at 30-month-old of Hanwoo steers. A total of 186 Hanwoo steers were used. Testosterone concentration, MAR, u-LMA and u-MAR according to grade were statistically significant differenced (p = 0.021, p < .0001, p = 0.003, p < .0001). Correlation analysis showed that MAR, u-LMA and u-MAR had a positive correlation (r = 0.921, 0.475, 0.760), and testosterone was †. This work was conducted with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project title: Expansion of ex-situ conservation of national major seed stocks and animal genetic resources for livestock industry development in Gyeongsangbuk-do, Project No. PJ011639052018) Rural Development Administration, Republic of Korea. Jae Jung Ha, Yun Sook Jung, Sung Hyun Kim and Jun Koo Yi contributed equally to this work. 1 Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. 2 Post-Doc., Department of Preventive Dentistry, Kyungpook National University, Daegu 41566, Korea. 3 Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. 4 Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. 5 Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. 6 Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. 7 Professor, Department of Statistics, Yeungnam University, Gyeongsan 38541, Korea. 8 Professor, Department of Animal Biotechnology, Kyungpook National University, Sangju 37224, Korea. 9 Professor, Department of Ecology and Environmental Science, Kyungpook National University, Sangju 37224, Korea. 10 Professor, China-US(Henan) Hormel Cancer Institute, No. 127 Dongming Road, Zhengzhou, Henan 450008, China. 11 Corresponding author: Agriculture researcher, Gyeongsangbuk-Do Livestock Research Institute, Yeongju 36052, Korea. E-mail: [email protected].

(2) 1722. J. Ha · Y. Jung · B. Kim · D. Jung · D. Oh · D. Kim · J. Lee · M. Kim · O. Kwon · S. Kim · J. Yi. a negative correlation (r = −0.278) between the grade. LMA was positive correlation (r = 0.218), and MAR had a negative correlation (r = −0.322) between the testosterone concentration. Carcass grade (LMA, BFT, MAR) and ultrasonic carcass grade (u-LMA, u-BFT, u-MAR) were positive correlation (r = 0.768, r = 0.691, r = 0.854). Keywords: Carcass grade, Hanwoo steers, testosterone concentration, ultrasonic measurement.. 1. Introduction Hanwoo (Korean native cattle) for meat has been improving continuously to select and raise cattle with excellent marbling, which suits the tastes preference of customers seeking Hanwoo beef (Lee, 2016). To improve the quality of Hanwoo, research institutes are estimating phenotypic breeding values, based on ultrasonic bio-diagnosis (Kim et al., 2012) and seeking methods of improving marbling and increasing farm revenue as well as, adjusting the shipping date (Choi et al., 2017) to produce optimal quality Hanwoo. In recent times, a variety of studies have been conducted, based on artificial feeding methods, including the use of additives. But there is still a shortage of studies on high quality meat production methods using concentrations and regulation of hormones in physiological metabolism of Hanwoo. According to the theoretical definitions of related studies, as a physiological organic matter, hormone is created in a specific cell and integrates and controls functions of each part of the body or of the whole by penetrating into other part of the same body or being transported (Shin, 2017). In general, hormones are divided into peptide hormones and steroid hormones. It has been reported that among these hormones, a steroid-affiliated sex hormone divides living organisms into females and males during the development phase and performs an important role in breeding and growth (Kang, 2011). In particular, estrogen, a female hormone engaging in breeding, testosterone, a male sex hormone, and progesterone, a corpus lutein hormone, are representative sex hormones. Among them, testosterone is a representative androgen secreted by a male testis and performs functions, such as expression of secondary sex characters, protein anabolism, and promotion of muscle growth (Hong et al., 2007). Also, it has been reported that sex hormones are involved in accumulation of additional fat (Kubo et al., 2008). As part of these functions, cell differentiation was inhibited when a mouse adipocyte was treated with testosterone, but on the other hand, estrogen promoted differentiation (Dieudonne et al., 2000). Kim et al. (2007)s research also reported that the application of testosterone influenced proliferation in the latter half of porcine preadipocyte culture, but then described that additional experiments were needed, since the result differed from the existing study results. Although research papers on sexual maturity in Hanwoo cows have been published recently (Jeong et al., 2016), there is an absence of data regarding steers and testosterone. Therefore, this study has been carried out in order to analyze testosterone levels in the blood of 30-month-old steers and prior ultrasonic biometric values for estimation of grades and to compare mutual statistical levels among the grading results of actually slaughtered steers, based on the preceding study findings..

(3) Statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic measurement1723. 2. Materials and methods Section 2.1 introduces animals and feeding management. Section 2.2 to 2.4 introduce experiment performance and data collecting methods and lastly, Section 2.5 introduces a statistical analysis method used in this study 2.1. Animals The animals used in the experiment were castrated when they turned 6 months old and were raised for 24 months to be fattened. Data were collected from 186 of 30-month-old Hanwoo that were scheduled to be slaughtered, in the livestock research institute for a year. The feed and feeding program of animals proceeded, on the basis of the Korean Feeding Standard for Hanwoo established by the National Institute of Animal Science in 2012. 2.2. Ultrasonic measurement Ultrasonic measurements were carried out on nearly the entire test animals immediately before they were shipped. To evaluate live carcass quality traits, 2 MHz real time ultrasonic equipment was applied between the 13th rib and lumber vertebra interface (HS-2000; HONDA, Japan). The images reflecting ultrasonic back fat thickness (u-BFT), longissimus muscle area (u-LMA) and marbling score (u-MS) were processed and analyzed using the Image-pro Express (Version 4.1, Media Cybernetics, USA) software. Animals without ultrasonic data were analyzed (Figure 2.1). This is a comparison of the meat quality between the slaughtered meat and the ultrasonic images just before the slaughter. The higher the quality level, the more difficult it is to read and observe ultrasonic images. 2.3. Carcass grade decision All of the steers were slaughtered at a local municipal slaughterhouse at 30 months of age, and carcass quality traits were evaluated according to the Korean carcass grading standard (KAPE, 2017). Carcasses were ribbed between the last rib and the first lumbar vertebra to determine back fat thickness (BFT), longissimus muscle area (LMA) and quality traits. Yield grade was determined by the cold carcass weight, adjusted back fat thickness and longissimus muscle area, and quality grade was determined by the marbling, meat color, fat color, texture and overall maturity score. Back fat thickness was determined over the medial third part of the longissimus muscle area. Marbling in the longissimus muscle area was scored from 1 (devoid) to 9 (abundant) according to the standard. Meat color was scored from 1 (brightly cherry red) to 7 (extremely dark red) and fat color was scored from 1 (white) to 7 (dark yellow) according to the standard. Firmness was water holding capacity and elasticity of the longissimus muscle area in the grade decision region, and was scored from 1 (firm) to 3 (soft) according to the reference index. Maturity was regarded as the ossification of the cartilage in the left semiconductor backbone thorn promontory and was scored from 1 (youthful) to 9 (mature). Yield index was determined by the following equation: Yield index = 68.184-(0.625×back fat thickness (mm))+(0.130×longissimus muscle area (cm2))(0.024×carcass weight (kg))+3.23 (for Hanwoo only). Yield grade was scored as follows. 3 = A grade (yield index≥67.20);.

(4) 1724. J. Ha · Y. Jung · B. Kim · D. Jung · D. Oh · D. Kim · J. Lee · M. Kim · O. Kwon · S. Kim · J. Yi. 2 = B grade (63.30≤yield index<67.20); 1 = C grade (yield index<63.30) Carcass quality grade was scored as follows. 5 = 1++ grade (marbling scored 8 or 9); 4 = 1+ grade (marbling scored 6 or 7); 3 = 1 grade (marbling scored 4 or 5); 2 = 2 grade (marbling scored 2 or 3); 1 = 3 grade (marbling scored 1). Figure 2.1 Ultrasonic analysis. 2.4. Testosterone analysis The analysis took place where the test animals were being reared. For collecting samples, about 10 cc of blood was drawn out of the Hanwoo jugular vein and it was centrifuged.

(5) Statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic measurement1725. in a SST evacuated tube to extract serums (Figure 2.2). After that, TESTO-CT2 (Cisbio, France) reagent was administered and the samples were analyzed using RIA method and DREAM GAMMA 10 (Shin-jin medics, Korea).. Figure 2.2 Experimental measurement. 2.5. Statistics analysis The collected data were analyzed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and the significance level for statistical significance was set at .05. We used non-parametric statistics analysis because data were not normally distributed. Testosterone concentration, longissimus muscle area (LMA), back-fat thickness (BFT), marbling score (MAR), ultrasonicmeasured LMA (u-LMA), BFT (u-BFT), and MAR (u-MAR) according to grade were analyzed by Kruskal-wallis test. Mann-whitney u test was used for post-hoc test if there was statistically significant difference between groups according to grade. Correlation between grade and testosterone concentration, LMA, BFT, MAR, u-LAM, u-BFT, and u-MAR was assessed by Spearman correlation method, which is a nonparametric method. The Spearman correlation formula is as follows. Pn. − R)(Si − S) qP , n 2 2 i=1 (Ri − R) i=1 (Si − S). δxy = qP n. i=1 (Ri. Ri =rank of xi ,R= mean rank of x, Si = rank of yi , R= mean rank of y.. 3. Results Section 3.1 presents the results of analyzing averages and standard deviations of testosterone, LMA, BFT, MAR, u-LMA, u-BFT and u-MAR depending on the grades of meat, and Section 3.2 considers the results of analyzing the correlations among these..

(6) 1726. J. Ha · Y. Jung · B. Kim · D. Jung · D. Oh · D. Kim · J. Lee · M. Kim · O. Kwon · S. Kim · J. Yi. 3.1. According to grade to the testosterone, LMA, BFT, MAR, u-LMA, u-BFT, u-MAR Testosterone concentration, MAR, u-LMA and u-MAR according to grade were statistically and significantly differed (p = 0.021, p < .0001, p = 0.003, p < .0001). The testosterone concentrations were 0.010.00 ng/ml in the 1++ grade group, 0.05±0.12 ng/ml in the 1+ grade group. 0.090.18 ng/ml in the 1 grade group, 0.48±0.98 ng/ml in the 2 grade group and 3.78±2.68 ng/ml in the 3 grade group. There was a lower testosterone concentration in the higher grade group (p = 0.021). u-LMA were 85.8±10.53 in the 1++ grade group, 81.63±13.33 in the 1+ grade group, 70.82±8.83 in the 1 grade group and 73.14±6.82 in the 2 grade group. There was higher u-LMA in the higher grade group (p = 0.003). u-MAR were 6.80±1.38 in the 1++ grade group, 4.95±1.64 in the 1+ grade group, 3.18±1.01in the 1 grade group and 2.43±0.79 in the 2 grade group. There was higher u-MAR in the higher grade group (p < 0.001) (Table 3.1, Figure 3.1). Table 3.1 According to grade to the testosterone, LMA, BFT, MAR, u-LMA, u-BFT, u-MAR N Testosterone LMA BFT MAR u-LMA u-BFT u-MAR (u-N) (N=186) (N=186) (N=186) (N=186) (N=153) (N=153) (N=153) Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Grade 0.34±1.06 87.26±10.84 11.44±4.93 4.79±2.16 77.83±12.10 8.68±3.88 4.40±1.93 1++ 24(23) 0.01±0.00c 93.27±8.65 9.91±3.53 8.18±0.40a 85.80±10.53a 7.60±3.20 6.80±1.38a 1+ 46(45) 0.05±0.12c 87.25±11.91 11.33±4.40 6.04±1.40b 81.63±13.33ab 9.32±3.89 4.95±1.64b 1 43(42) 0.09±0.18c 84.95±9.89 12.76±5.68 4.52±0.51c 70.82±8.83c 8.83±4.26 3.18±1.01c 2 44(43) 0.48±0.98b 87.14±10.26 12.05±5.17 2.68±0.48d 73.14±6.82c 9.26±4.15 2.43±0.79cd 3 29(0) 3.78±2.68a 83.50±16.11 6.00±1.41 1.00±0.00e p-value 0.021 0.162 0.055 < .0001 0.003 0.703 < .0001 P-values by Kruskal wallis test a,b Different characters mean significant difference between groups by Mann-whitney u test. 3.2. Correlation between grade and testosterone concentration, LMA, BFT, MAR, u-LAM, u-BFT, and u-MAR Correlation analysis showed that MAR, u-LMA and u-MAR had positive correlation (r = 0.921, 0.475, 0.760), and testosterone had a negative correlation (r = −0.278) between the grade. LMA had positive correlation (r = 0.218), and MAR had a negative correlation (r = −0.322) between the testosterone concentration. Therefore, the higher the grade, the lower the testosterone concentration, and the higher the MAR, u-LMA and u-MAR were detected. The correlations with the grade were in the order of MAR, u-MAR, u-LMA, and testosterone. Carcass grade (LMA, BFT, MAR) and ultrasonic carcass grade (u-LMA, uBFT, u-MAR) were positive correlation (r = 0.768, r = 0.691, r = 0.854) (Table 3.2).. 4. Discussion This study was performed to compare and analyze the effect of concentrations of testosterone, a steroid-affiliated male sex hormone, on carcass grades of steers, and the accuracies of ultrasound-measured carcass grades. Marbling and longissimus muscle area have the biggest influence on a carcass grade which is an important scale for high profitability in Hanwoo.

(7) Statistical effect analysis on the testosterone concentration, carcass grade and ultrasonic measurement1727. Figure 3.1 According to grade to the MAR, testosterone, u-LMA, u-MAR. breeding (KCSAB, 2009). Like Snyder et al. (2000)s research reporting that among sex hormones related to growth of muscles and fatty deposits, testosterone increased muscles, but decreased fatty tissues when it was administered to men. The Singh (2006)s research demonstrated that adipocyte differentiation was inhibited when 3T3-L1 was treated with testosterone, and the same patterns (0.218∗ , -0.322∗∗ ) were found in this study. The estimation of grades using ultrasonic diagnosis has been relying on inspectors skill and visual analysis. It has been reported that the accuracy is below 50% on average (Cheong et al., 2012). However, when comparing and analyzing the correlations among the estimated carcass grades and the real carcass grades using ultrasonic measurement right before slaughter to identify the researchers ultrasonic diagnosis capability and the level of reliability, LMA, BFT, and MAR had a very significant accuracy of 0.786∗∗ , 0.691∗ , and 0.854∗∗ respectively. This implies statistical levels of ultrasonic diagnosis and testosterone concentrations could be accurate to some degree. So far, ultrasonic measurement has been dependent on non-.

(8) 1728. J. Ha · Y. Jung · B. Kim · D. Jung · D. Oh · D. Kim · J. Lee · M. Kim · O. Kwon · S. Kim · J. Yi. Table 3.2 Correlation between grade and testosterone concentration, LMA, BFT, MAR, u-LAM, u-BFT, Grade Grade 1.000 Testosterone -0.278∗∗ LMA 0.193 BFT 0.009 MAR 0.921∗∗ u-LMA 0.475∗∗ u-BFT -0.048 u-MAR 0.760∗∗ ∗∗ p < 0.01, ∗ p < 0.05. Testosterone 1.000 0.218∗ -0.138 -0.322∗∗ 0.038 -0.003 0.093. and u-MAR LMA BFT. 1.000 -0.081 0.278∗ 0.786∗∗ -0.178 0.599∗∗. 1.000 0.034 -0.114 0.691∗ -0.214. MAR. u-LMA. u-BFT. u-MAR. 1.000 0.509∗∗ -0.033 0.854∗∗. 1.000 0.167 0.724∗∗. 1.000 0.034. 1.000. professionals visual analysis and also depended on each livestock farming area, unfortunately it has been impossible to trust in estimated carcass grades due to the low accuracy (Park et al., 2012). This problem has brought about frequent income disputes between industries and livestock farms (Lee and Lee, 2016). To alleviate this issue, this study result would need to be used. Moreover, it is supposed that in order to predict marbling and grades for Hanwoo while they are alive, constantly analyzing testosterone concentrations in the blood of steers and adjusting the overall shipping date, based on experts ultrasound-measured values would create positive influence on the improvement of farm revenue in the future by creating a relatively reliable carcass grades.. References Cheong, J. K., Oh, Y. T., Choi, H. N., Lee, C. H., Kim, K. H., Kim, K. Y., Choi, Y. H., Kim, H. C. and Hwang, J. M. (2012). Effects of geographic locations and year-seasons of birth on ultrasound scanned measures and carcass traits of Hanwoo steers. Journal of Animal Science and Technology, 54, 247-253. Choi, T. J., Choi, Y. H., Park, B. H., Cho, G. H., Alam, M., Kang H. Y., Lee S. S. and Lee, J. G. (2017). Study on the application of ultrasound traits as selection trait in Hanwoo. Journal of Agriculture & Life Science, 51, 117-126. Dieudonne, M. N., Pecquery, R., Leneveu, M. C. and Giudiecelli, Y. (2000). Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: Evidence for sex and siterelated specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisomes proliferator-activated receptor γ 2 . Endocrinology, 141, 649-656. Hong, L., Colpan, A., Peptan, I. A., Daw, J., George, A. and Evans, C. A. (2007). 17-Betaestradiol enhances osteogenic and adipogenic differentiation of human adipose-derived stromal cells. Tissue Engineering, 13, 1197-1203. Jeong, C. S., Jang, S. S., Lee, S. S. and Moon, Y. H. (2016). Effects of concentrate feeding level after sexual maturity on the growth and breeding performances, blood composition, and ultrasonic carcass traits in Hanwoo cows. Journal of Agriculture & Life Science, 50, 113-125. Kang, Y. H. (2011). Encyclopedia of life science, Academy Publishers, Seoul. KAPE (Korea institute for animal products quality evaluation) (2017). Korean carcass grading standard, Available online at http://www.ekape.or.kr/view/user/institution/intro.asp KCSAB (Korean cattle self-help cost administration board) (2009). Korean cattle consulting guidelines, Journal of Animal Science and Technology Press, Seoul. Kim, H. C., Lee, S. H., Dang, C. G., Jeon, G. J., Yeon, S. H., Cho, Y. M., Lee, S. M., Yang, B. S. and Kim, J. B. (2012). Effect of genetic parameters for ultrasound and carcass traits in Hanwoo. Journal of Animal Science and Technology, 54, 331-336. Kim, H. R., Lee, K. H., Choi, I. H. and Chung, C. S. (2007). Effect of sex steroid hormones on differentiation of pig preadipocytes. Journal of Animal Science and Technology, 49, 593-598..

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