Estrogenic Activity in the Meat of Broilers Treated with Estrogenes Hiroshi WADA, Akinao TANIMOTO, Akira KAWASAKI* and Narioki SEKIBA** (Okayama University School of Agriculture, *Okayama Prefectural Poultry Experiment Station, and **Fukuda Poultry Institute, Okayama, Japan) (Received for publication on December 1, 1962) In the recent years, the broiler industry has developed rapidly in Japan. In rearing broilers, synthetic estrogens have been used for the purpose of improving the growth rate and the meat quality of chicken. Diethylstilbestrol and hexestrol are commonly used as strogenic substances for this purpose. The problem of possible residue of estrogen in the meat of chickens reared by administration of such substances is very important from the viewpoint of public health. Chemical and biological assays have been developed for estrogenic activity of carcasses of birds and cattle treated with estrogen. Several reports were made on estrogen residue in the tissues of chickens treated with estrogen2～4,7). The present study is concerned with the residue of estrogen in the meat of broiler chickens treated with synthetic estrogens by three means of administration. Materials and Methods 1. Administration of estrogen to broiler chickens. Male broilers randomly selected from those administered with estrogens by the following three means were used in this study. They were fed commercial all-mashed feed. The hormone treatment was started at an age of 32 to 42 days. a. Injection: Crossbred chickens between the White Cornish and New Hampshire breeds or those between the White Plymouth Rock and New Hampshire breeds were used in this experiment. An injection (a product of Toshiba Pharmaceutical Co., Ltd.) was used as an estrogen. One milliliter of the injection contained 30mg of diethylstilbestrol (DS) and 6 mg of methyl thiouracil. Each bird was injected intramuscularly at the breast with a single dose of 0.5ml of the solution on the first day of experimint. These chickens were sacrificed at 77 days of age for the assay of estrogenicity of their meat. b. Implantation: The chickens used were the same crossbred as mentioned above. Two kinds of estrogen pellets (prepared by the company cited above) were used in this experiment. One of them contained 7.5mg of DS and 7.5mg of hexestrol, and the other 15mg of hexestrol. Each chicken was implanted subcutaneously just behind the comb with a single pellet on the first day of experiment and sacrificed 35 or 45 days following the implantation. c. Oral administration:"Euvestin Powder", premix of synthetic estrogens prepared by the Takeda Pharmaceutical Ind., Ltd., was used as estrogen. It was mixed in the feed at a rate of 0.2%. One gram of the premix contained 4.5mg of DS and 0.5mg of dicarboethoxystilbestrol (or Euvestin). Therefore, the total amount of synthetic estrogens added per kg of feed was 10mg. Chickens of four-way crossbred of the White Leghorn, Sussex, White Cornish, and New Hampshire breeds were fed the feed containing the premix powder during a period from 35 to 78 days of age, and sacrificed 2 days later. Jap. J. Zootech. Sci.. 34.(3) 201 1963.8. WADA・TANIMOTO・KAWASAKI・SEKIBA 2. Bioassay method of estrogen in meat. At the sacrifice of chickens, edible meat was removed. The fresh meat was ground and mixed with a commercial mouse feed at a ratio of 3.5: 1.0, so that the weight ratio of meat to feed might be approximately equal in air-dried state. The mixture of meat and feed was made into a form of pellet and then dried at 50℃ for two days. Estrogenic activity was assayed by examining the response of the uterus of immature mouse to the hormone. Generally, eight to ten mice were used for one assay. Immature mice, weighing 10 to 12g initially, were given 2g of the test meat-feed pellet per mouse daily for one week. In this method, mice were fed pellets of the control feed ad libitum after finishing the test pellet given every morning. On the 8th day, they were sacrificed. The uterue was removed from each of them free of mesentery, blotted on bibulous paper and weighed. Uterine weight was expressed as weight per 12g of body weight. Estrogenic activity was estimated from the uterine weight in relation to the standard curve of uterine response to estrogen, that is to be mentioned below. Standard curve of response of mouse uterus to diethylstilbestrol: The fresh meat of untreated male chickens was cut into small pieces, dried and ground. The ground meat was mixed in half with the same commercial mouse feed as stated previously. This was the control mixture of meat and feed, and made into pellets. On the other hand, graded amounts, varying from 0.25 to 10μg, of DS were added to 100g of the meat-feed mixture and made into a form of pellet weighing approximately 2g in air-dried state. Mice of the assay group were fed the hormone-containing pellets at a rate of 1g per mouse daily for 7 days. They were given control pellets every day after they had finished test pellets. On the 8th day, they were sacrificed. Their uteri were weighed in the same manner as described above. The average uterine weights per 12g of body weight were plotted as a curve against the dose of the estrogen. In this way was obtained the standard curve of uterine response to the dose of DS, on which determinations were based in this study. Results and Discussion The data used for the standard curve of response of mouse uterus to DS are shown in Table 1. Results of the assay of residual estrogen in the meat of broilers treated with the hormone are given in Table 2. Table 1. Effect of graded doses of diethylstilbestrol (DS) addet to control meat-feed mixture 202 Recidual estrogen in meat of broiler Table 2. Assays of estrogen residue in chickin meat with the uterine response of immature female mice * DS: diethylstilbestrol, and EV: Euvestin, a proprietary name of p, p'-dicarboethoxy-oxy- trans-α, β-diethylstilbene. ** Given 7.5mg DS and 7.5mg hexestrol. † H: New Hampshire L: White Leghorn C: White Cornish R: White Road S: Sussex The average uterine weight of mice fed pellets of the meat-feed mixture to be tested is to indicate the presence of estrogen equivalent to the amount of DS corresponding to the uterine weight shown in Table 1 in 100g of dried meat. The uterine weight per 12g of body weight was in a range from 8.5 to 12.9mg, averaging 11.9±0.6mg, in mice fed pellets of the control meal-feed mixture. The uterine weight per 12g of body weight was in a range of 7.3±0.2 to 11.9±1.1mg, averaging 10.1±0.7mg, in mice fed the meat of chickens which had been injected with DS and methyl thiouracil. The average uterine weighg per 12g of body weight was 8.7±0.1mg, in a range of 7.8±0.5 to 9.4±0.9mg, in mice fed the meat of estrogen-implanted broilers. And the uterine weight per 12g of body weight in mice fed the meat of broilers given DS orally averaged 10.0±0.3mg with range of 8.5±0.4 to 11.9±0.5mg. Thus, the mean uterine weight of mice fed the meat of estrogen-treated chickens did not exceed the control value. Therefore, in the experimental conditions of this study, estrogen residue may be thought negative in the meat of chickens treated with synthetic estrogen. 203 WADA・TANIMOTO・KAWASAKI・SEKIBA Although a graded response was observed in uterine weight as the amount of added DS increased, previous investigators noticed that control animals fed commercial mouse feed had larger uteri than those fed DS in non-ovariectomized weanling mice1,7). The present authors used intact immature mice weighing 10 to 12g each, while the investigators mentioned above employed mice weighing 13.5 to 19.4g each. Regularly garded responses were obtained with the increase in amount of added estrogen. No undesirable increase in weight of the uterus of the control mouse over that of the mouse fed estrogen was observed. Therefore, in the present study, non-ovariectomized mice weighing 10 to 12 each at the start of assay were used exclusively. The actual sensitivity of this method was more than 2 parts per billion. JONES and DEATIERAGE4)observed no significant amount of DS in the liver and breast muscle plus abdominal fat of the chicken which had received 15mg of DS in a form of fat-type pellet or aqueous pasty suspension about 4.5 weeks following the treatment. SWIFT5) detected no residue of estrogen in the muscle of chickens, but a little in the extracts of the liver and skin of chickens slaughtered 60 or 90 days after the treatment. STOB et al.2) reported that the rate of absorption of a pellet, containing 12mg of stilbestrol, implanted subcutaneously in the neck of a chicken decreased gradually, the mean daily amount of absorbed hormone being 486μg in the first week and 225μg in 4 weeks following the treatment, and that residual estrogen per gram of dried meat was 0.05 and 0.01μg at 7 and 28 days, respectively, after the implantation. MERKERet al.7), who studied the estrogenic activity of free and conjugated phenolic residues of the carcasses of chickens treated with 12mg of DS pellet, reported that both phenolic residues and air-dried meat failed to reveal any significant estrogenic activity when tested by the oral-feeding method of STOB. In the carcasses of beef cattle treated with synthetic estrogen, STOB et al.2,9) detected residues of estrogen.