Peripartal propylene glycol supplementation and metabolism, animal health, fertility, and production in dairy cows.

The effect of peripartal supplementation with concentrate enriched at 10% propylene glycol (PG) on metabolism, animal health, fertility, and milk production was studied using 234 cows from 8 dairy farms with production averages of 8019 to 10,656 kg/yr. The feeding schedule for the PG group (n=117) was as follows: 13 d antepartum: 1.5 kg/d (= 150 mL PG); 12 d antepartum until parturition: 3 kg/d (= 300 mL PG); 1 to 12 d postpartum: 1 kg/d (= 100 mL PG). Control cows (n=117) received the same concentrate without PG. From a subset of cows (PG: n = 43; control: n = 40), blood samples were collected at 6, 3, and 1 wk antepartum, 3 d antepartum, on the day of parturition, and 1, 3, 5, 7, 9, and 11 wk postpartum for the determination of nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), insulin-like growth factor (IGF)-I, and activities of aspartate-aminotransferase (AST) and glutamate-dehydrogenase (GLDH). From another subset of cows (PG: n=11; control: n=10), blood samples were collected 1 wk antepartum, on the day of parturition, and 1 wk postpartum to determine immunophenotypical and functional parameters of blood neutrophils. From 1 wk antepartum to 1 d postpartum, concentrations of NEFA were significantly lower in cows receiving PG compared with controls. Also, concentrations of BHBA in cows receiving PG were significantly lower from 1 wk antepartum until 7 wk postpartum. Concentrations of IGF-I were significantly higher in the PG group, from 1 wk antepartum until 1 wk postpartum than in the control group. Activities of AST and GLDH did not differ between groups. Immunophenotypical and functional characteristics of blood neutrophils were not influenced by treatment nor were animal health, reproduction, or milk production. Although indicators of metabolic status were improved by peripartal use of PG-enriched concentrate, economic benefits are questionable for dairy farms with good nutritional programs, as economically important factors such as milk production, animal health, and fertility were not influenced.

Quantitative assessment of foetal exposure to trenbolone acetate, zeranol and melengestrol acetate, following maternal dosing in rabbits.

1. Residues of commonly used growth-promoting agents found in animal meat can be hormonally active and they have been implicated as possible endocrine disruptors in man. Although these compounds could be potentially detrimental to the developing foetus, it is not clear whether and to what extent they pass through placental barrier. 2. This issue was addressed using the rabbit as an animal model. Pregnant rabbits were treated with trenbolone acetate, zeranol or melengestrol acetate beginning at gestation day 14. Levels of active substances in plasma were screened by means of specific ELISA systems. The residues of parent compounds and their metabolites were quantified in maternal and foetal tissues on gestation day 27 using validated, sensitive HPLC/ELISA methods. 3. All three compounds crossed the placental barrier and were detectable in foetal tissues. The extent of tissue concentration varied depending on the compound and tissue analysed. Gender differences were observed in some instances.

Effects of melengestrol acetate on reproductive behavior and concentrations of LH and testosterone in bulls.

We investigated the use of an orally active progestin (melengestrol acetate; MGA) to suppress reproductive activity in yearling beef bulls. Twenty-four crossbred bull calves were given a daily dose of 0, 0.5, 1.0, or 2.0 mg MGA for 99 d. Pulsatile patterns of LH and concentrations of testosterone and MGA were characterized on d 8, 36, 63, and 92 of the experiment. Numbers of aborted mounts, mounts with intromission, total mounts, and flehmen responses were assessed on d 15, 43, 71, and 99. Plasma concentrations of MGA were proportional to dose of MGA. Melengestrol acetate did not consistently affect mounting behavior in a dose-related manner, but, on d 99, number of total mounts for MGA-treated bulls was lower (P = 0.07) than that for control bulls. On d 15, MGA suppressed (P = 0.07) numbers of flehmen responses in a dose-dependent manner, but this effect was not sustained throughout the experiment. On d 8, concentrations of testosterone in control bulls were higher (P = 0.02) than in MGA-treated bulls, but this effect was not observed at other time periods. Overall, MGA caused slight decreases in mean concentrations of LH (P = 0.09) and LH pulse frequency (P = 0.06). Scrotal circumference was not affected by MGA. None of the behavioral traits was correlated with mean concentrations of LH or LH pulse frequency. Mounting activity was not correlated with testosterone concentrations, but number of flehmen responses was positively correlated with testosterone concentrations (P = 0.01). These results fail to support the hypothesis that progestins impair male sexual behavior or fertility in males.

Effects of synthetic progestagens on the mRNA expression of androgen receptor, progesterone receptor, oestrogen receptor alpha and beta, insulin-like growth factor-1 (IGF-1) and IGF-1 receptor in heifer tissues.

Synthetic progestagens like melengestrol acetate (MGA) are widely used for oestrus synchronization and for growth promotion in cattle production. The metabolic effects exceed its primary potency as a progestagen. It is speculated that MGA stimulates follicle development and thereby endogenous oestrogen production, but inhibits ovulation. To investigate the dose-dependent effects on mRNA expression levels, six heifers were fed for 8 weeks with different levels of MGA (0.5, 1.5, 5 mg) daily and two heifers served as controls. The expression of steroid receptor mRNA [androgen receptor (AR), progesterone receptor (PR), oestrogen receptor (ER) ERalpha and ERbeta], insulin-like growth factor-1 (IGF-1) and its receptor were quantified in liver, neck (m. splenius) and shoulder muscularity (m. deltoideus). Plasma concentrations of IGF-1 were quantified by radioimmunoassay. In treated animals the MGA plasma levels were elevated over the complete treatment period, corresponding to the MGA treatment concentrations. IGF-1 concentrations of control animals were at constant levels. Plasma levels for oestradiol (E2) and IGF-1 were increased in the low MGA treatment group. Overdosed MGA decreased progesterone (P4) and E2 levels. To quantify the IGF-1 and all receptor mRNA transcripts, sensitive and reliable real-time reverse transcription-polymerase chain reaction (RT-PCR) quantification methods were developed and validated in the LightCycler. A dose-dependent relationship between increasing MGA concentration and mRNA expression was observed in liver for AR and IGF-1 receptor, and in neck muscularity for IGF-1. ERalpha in liver and neck muscle showed a trend of increasing expression.

The fate of trenbolone acetate and melengestrol acetate after application as growth promoters in cattle: environmental studies.

The steroids trenbolone acetate (TbA) and melengestrol acetate (MGA) are licensed as growth promoters for farm animals in several meat-exporting countries. Although many studies have explored their safety for both animals and consumers, little is known about their fate after excretion by the animal. Our study aimed to determine the residues and degradation of trenbolone and MGA in solid dung, liquid manure, and soil. In animal experiments lasting 8 weeks, cattle were treated with TbA and MGA. Solid dung and, in case of trenbolone, liquid manure were collected and spread on maize fields after 4.5 and 5.5 months of storage, respectively. Determination of the hormone residues in all samples included extraction, clean-up (solid-phase extraction), separation of metabolites and interfering substances by HPLC (RP-18), and quantification by sensitive enzyme immunoassay. Procedures were validated by mass spectrometry (MS) methods. During storage of liquid manure the level of trenbolone decreased from 1,700 to 1,100 pg/g (17alpha-isomer), corresponding to a half-life of 267 days. Before storage, the concentrations in the dung hill ranged from 5 to 75 ng/g TbOH and from 0.3 to 8 ng/g MGA. After storage, levels up to 10 ng/g trenbolone, and 6 ng/g MGA were detected. In the soil samples trenbolone was traceable up to 8 weeks after fertilization, and MGA was detected even until the end of the cultivation period. The results show that these substances should be investigated further concerning their potential endocrine-disrupting activity in agricultural ecosystems.

Studies on the antibody response of Lama glama--evaluation of the binding capacity of different IgG subtypes in ELISAs for clenbuterol and BSA.

Camelidae are known to produce three subtypes of immunoglobulin G (IgG), two of which are devoid of light chains. Two llamas (Lama glama) were immunised against clenbuterol-bovine serum albumin (BSA). Enzyme-linked immunosorbent assays (ELISAs) for clenbuterol and BSA on the basis of protein A-coated microtitration plates were established to investigate the titre development. Three subclasses of IgG (IgG(1): 29+66KDD, IgG(2): 52KDD, IgG(3): 56KDD) depending on their different binding properties to protein A and protein G could be separated chromatographically. Only IgG(1), which consists of conventional four-chain antibodies, bound to clenbuterol, whereas all forms of heavy-chain antibodies merely bound BSA.

Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER beta mRNA with real-time RT-PCR.

We have examined the tissue-specific mRNA expression of ER alpha and ER beta in various bovine tissues using real-time RT-PCR. The goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue-specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (alpha-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyperestrogenism, in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple-dose implantations (0x, 1x, 3x, 10x) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ER alpha and ER beta transcripts also in low-abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissues exhibited a specific ER alpha and ER beta expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ER alpha mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis that ER beta may have different biological functions than ER alpha, especially in kidney and jejunum.

Possible health impact of animal oestrogens in food.

Oestrogens govern reproductive functions in vertebrates, and are present in all animal tissues. The theoretical maximum daily intake (TMDI) of oestradiol-17beta by consumption of cattle meat is calculated to be 4.3 ng. Following the use of oestradiol-containing growth-promoting agents, TMDI is increased by a factor of 4.6 to 20 ng oestradiol-17beta, assuming that single dosage and 'good animal husbandry' are observed. Pork and poultry probably contain similar amounts of oestrogens as untreated cattle. The mean concentration of oestradiol-17beta in whole milk is estimated at 6.4 pg/ml. Scarce data available on eggs report up to 200 pg/g oestradiol-17beta. The risk evaluation of oestrogenic growth-promoting agents is limited by analytical uncertainties. Residues of oestradiol-17alpha and the importance of oestrogen conjugates are widely unknown. The performance of mass spectrometry still needs to be improved for confirmation of oestrogen concentrations in most food. At present, the potential relevance of oestradiol acyl esters, the actual daily production rate of oestradiol in prepubertal children, and the role of oestradiol metabolites in cancer are obscure. The presence of different cytoplasmic oestrogen receptor subtypes and potential oestradiol effects in non-reproductive functions require further examination.

Possible health impact of phytoestrogens and xenoestrogens in food.

Plants produce estrogen-like substances, denominated phytoestrogens, which are present in many human foodstuffs. The consumption of phytoestrogens has been associated with a variety of protective effects. Their relative estrogenic potency combined with their concentrations in food and human plasma indicate biological relevance. However, their biological properties differ from those of estradiol or other endogenous estrogens in humans. For instance, their possible effects on SHBG, inhibition of steroid metabolizing enzymes, anti-proliferative and anti-angiogenetic and other side effects have been described. Furthermore, phytoestrogens can exert estrogenic and antiestrogenic activities at the same time and their potency and metabolism have not been yet elucidated in all cases. In recent decades growing evidence has accumulated on the hormone-like effects of synthetic chemicals that appeared in the environment. The possible impact of xenoestrogens, to which humans are also exposed through the food chain, needs to be further clarified as well. The molecular effects and control mechanisms of these substances, their pharmacokinetics, threshold levels and dose-response differences are issues that require further research before a full assessment of their effect on humans can be drawn. Evaluating the total exposure and impact of this estrogenic effect is very challenging because of the lack of specific knowledge in some areas and the differences in the biological activity among these substances, as pinpointed in this review.

A sensitive enzyme immunoassay (EIA) for the determination of melengestrol acetate (MGA) in adipose and muscle tissues.

The development of a sensitive screening method of MGA residues in bovine perirenal fat and muscle based on a competitive microtitration plate enzyme immunoassay is described. The samples were extracted with petroleum ether and purified with octadecyl-silica-cartridges. The detection limit for fat was 0.4 ng/g andfor muscle tissue 0.05 ng/g, much lower than requiredfor reliable detection of positive samples. The mean recovery rates of fortified samples amount to 75%, the mean intraassay variations to 7% and the interassay variation to 13%. Determination limits were validated for fat at 2 ng/g and for muscle at 0.1 ng/g. The efficiency of the new screening method was successfully demonstrated by the direct comparison to GC-MS and LC-MS methods performed at natural positive samples originating from an animal experiment in which the labelled dose (0.5 mg per animal and day) with and without a 48 h withdrawal period or 3-fold or 10-fold the amount of MGA, respectively, was fed to Holstein Frisian heifers. In conclusion, this new screening method can be used for sensitive determination of MGA residues in adipose tissues even after low treatment doses or longer withdrawal periods.

Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.

Certain hormonal growth promoters are licensed in several beef producing countries outside the European Union (EU). Use in compliance with Good Veterinary Practice is mandatory. As risk assessment of hormone residues in animal tissues up to now has neglected potential off-label use, the present study dealt with two topics: 1) multiple treatment with the implant preparations Finaplix-H (200 mg trenbolone acetate), Ralgro (36 mg zeranol) and Synovex-H (200 mg testosterone propionate plus 20 mg estradiol benzoate) in heifers (1-fold, 3-fold and 10-fold dose), and 2) non-approved treatment of female veal calves (1-fold dose of Synovex-H or Synovex Plus with 200 mg trenbolone acetate plus 28 mg estradiol benzoate). Residues of estradiol-17beta, estradiol-17alpha, estrone and testosterone, trenbolone-17beta, trenbolone-17alpha and trendione or zeranol, respectively, were measured in loin, liver, kidney and peri-renal fat by high performance liquid chromatography/enzyme immunoassay (HPLC/EIA) after liquid-liquid extraction and solid-phase clean-up. The hormone residues in the multiple-dose experiments were dose-dependent and partially exceeded the threshold values: in the liver in one animal after 3-fold dose and in two animals after 10-fold dose of Finaplix-H, and in the liver and kidney after 3-fold and 10-fold dose of Synovex-H. Mean hormone residues in calves were mainly below those of heifers and did not infringe threshold values.

Detection of anabolic residues in misplaced implantation sites in cattle.

Eight weeks before slaughter, 26 heifers, 2 calves, and 1 steer were implanted with licensed anabolic preparations at off-label injection sites. After slaughter, 24 of 31 implantation sites (77%) were detected. Residual pellets of Revalor H contained a mean of 42.9 mg trenbolone acetate (range 19.8-57.7 mg) and 4.6 mg (1.96-6.45 mg) estradiol, corresponding to 30% (19.8-57.7%) and 32.7% (14.0-46.6%) of the originally applied dose, respectively. In the tissue areas containing residual Revalor H pellets, total residues ranged from 14.8 microg to 12.6 mg trenbolone acetate, 41.7 microg to 1.45 mg trenbolone, and 11.1 microg to 3.39 mg estradiol. The outer tissue areas of the injection sites contained <2 microg hormones. The preparations Synovex H, Finaplix H, Implus S, and Component EC behaved similarly to Revalor H. Residues of Synovex Plus were low, whereas the Compudose silicone rubber contained 58.8% of the implanted dose, but left no significant tissue residues. If implantation sites are processed in meat manufacturing, international threshold levels of the respective substances will be exceeded in tons of meat products.

Dose-dependent effects of melengestrol acetate (MGA) on plasma levels of estradiol, progesterone and luteinizing hormone in cycling heifers and influences on oestrogen residues in edible tissues.

Melengestrol acetat (MGA) is widely used as a growth promoting feed additive in cattle breeding in the USA and several other non-European countries. To explore the physiological effects of MGA four heifers were fed during 8 weeks with 0.5 mg MGA daily as registered in the USA and two heifers each received 0, 1.5 or 5 mg/day, respectively. Plasma samples were collected twice a week and concentrations of MGA, progesterone (P4) and estradiol-17beta (E2-17beta) were quantified. The pulsatile secretion of luteinizing hormone (LH) was investigated in 6-hour profiles before and during treatment. After slaughter the reproductive organs were examined and oestrogen residues in edible tissues were measured. Four days after the beginning of MGA feeding MGA concentrations in plasma reached levels of 30 and 100-400 pg/mL depending on the dose received. Three weeks after the beginning of MGA feeding P4 plasma concentrations had dropped to base levels below 0.3 ng/mL in all three treatment groups. Mean plasma E2-17beta levels increased in physiological range from 1 to 5 pg/mL during 0.5 mg MGA/day feeding with many acyclic peaks. Overdosed MGA decreased E2 levels and suppressed cyclic peaks. Number and size of ovarian follicles were not altered by any treatment. Mean LH levels and pulse frequencies increased significantly during labelled treatment (0.5 mg/day), while higher doses had reducing effects. The development of corpus luteum was suppressed. E2-17beta residues in fat increased about 300% following labelled MGA treatment.

Characterisation of the affinity of different anabolics and synthetic hormones to the human androgen receptor, human sex hormone binding globulin and to the bovine progestin receptor.

For the steroidal growth promoters trenbolone acetate (TBA) and melengestrol acetate (MGA) neither the complete spectrum of biological activities nor the potential endocrine disrupting activity of their excreted metabolites in the environment is fully understood. The potency of these substances in [3H]dihydrotestosterone ([3H]-DHT) displacement from the recombinant human androgen receptor (rhAR) and from human sex-hormone binding globulin (hSHBG) was evaluated. In addition, the potency for [3H]-ORG2058 displacement from the bovine uterine progestin receptor (bPR) was tested. For comparison, different anabolics and synthetic hormones were also tested for their binding affinities. For 17beta-trenbolone (17beta-TbOH), the active compound after TBA administration, an affinity the rhAR similar to dihydrotestosterone (DHT) and a slightly higher affinity to the bPR than progesterone were demonstrated. The affinity of the two major metabolites, 17alpha-trenbolone and trendione, was reduced to less than 5% of the 17beta-TbOH-value. The affinity of these three compounds and of MGA to the hSHBG was much lower compared with DHT. MGA showed a 5.3-fold higher affinity than progesterone to the bPR but only a weak affinity to the rhAR. The major MGA metabolites have an affinity to the bPR between 85% and 28% of the affinity of progesterone. In consequence, MGA and TBA metabolites may be hormonally active substances, which will be present in edible tissues and in manure. We conclude that detailed investigations on biodegradation, distribution and bio-efficacy of these substances are necessary.

Detection of melengestrol acetate residues in plasma and edible tissues of heifers.

The aim of this study was to gain knowledge of residue formation after the use of melengestrol acetate (MGA) as a growth-promoting agent. Two Holstein-Friesian heifers each received a daily dose through the feed of 0, 0.5 mg (2 heifers with and without withdrawal each), 1.5 mg or 5.0 mg MGA for 8 weeks. MGA residues in plasma were screened by enzyme immuno-assay (EIA). Concentrations in kidney, liver, and muscle were quantified by liquid-chromatography-mass spectrometry (LC-MS), and in fat by gas chromatography-mass spectrometry (GC-MS). MGA levels in plasma were 40, 128, and 280 ng/L, respectively. Residues accumulated in muscle and kidney (5-fold), liver (20-to-40-fold), and fat (200-fold). After administration of 1.5 mg per day the mean MGA concentration in fat was 29 micrograms/kg and thus violated USA regulations which specify a limit of 25 ppb. Therefore the labelled use of MGA (0.5 mg per day) has to be officially controlled.

Increased milk levels of insulin-like growth factor 1 (IGF-1) for the identification of bovine somatotropin (bST) treated cows.

The present EU moratorium banning the use of bST to increase milk yield implies the need for official controls. Our study aimed to identify milk from bST treated cows via the induced increase of insulin-like growth factor 1 (IGF-1) concentrations. A non-extraction radioimmunoassay for IGF-1 was improved and thoroughly validated for milk. Accuracy was 99% recovery in a fortified sample material, the precision was 5.1% intra-assay variation and 13.4% inter-assay variation. Parallelism was proved by a dilution experiment which yielded a regression line with a slope (-0.7%) not significantly different from zero (P = 0.534). Naturally occurring milk IGF-1 levels were recorded in 5777 random milk samples from the Bavarian dairy cow population. In samples from lactation week 7 to 33, the effect of somatic cell count (SCC), protein content and parity could be quantified and corrected; thus a normal distribution (-0.068 mean +/- 0.440 s) of the corrected logarithmic IGF-1 levels (corr ln IGF-1) was obtained. IGF-1 concentrations occurring in milk from bST treated cows were recorded in 33 Brown Swiss cows treated once with rbST (POSILAC). Mean corr in IGF-1 levels increased by 0.828 and 0.477 in first parity and older cows, respectively. Thus 60% and 29%, respectively, of the positives could be detected at a 95% probability. If our results are confirmed in experiments with more bST treated cows and with prolonged treatment intervals. IGF-1 measurements might be useful to monitor for bST application in milk samples.

Human insulin-like growth factor I (IGF-I) produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic activity, and effects on IGF-binding proteins.

Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions offering a range of important therapeutic applications. To evaluate a system for large-scale production of this peptide in the mammary glands of transgenic livestock, we generated transgenic rabbits carrying fusion genes in which a synthetic DNA coding for human IGF-I (hIGF-I) was placed under the transcriptional control of regulatory elements isolated from the bovine alpha S1-casein (alpha S1-cas) gene. Western blot analysis of milk from alpha S1-cas-hIGF-I transgenic rabbits demonstrated production of high amounts of mature hIGF-I peptide (7.6 kDa). Quantitative analysis by RIA revealed hIGF-I levels between 50 and 300 micrograms/ml milk. Recombinant hIGF-I purified from the milk of alpha S1-cas-hIGF-I transgenic rabbits bound to IGF-I receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I produced in Escherichia coli. Ligand blot analysis of milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa IGF-binding proteins (IGFBPs). The 30-kDa IGFBP was shown to be IGFBP-2 by immunoprecipitation using an antiserum raised against human IGFBP-2. Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction in alpha S1-cas-hIGF-I transgenic rabbits. The latter displayed slightly increased milk yield, but no significant changes in total protein content or overall milk protein composition, and reared their offspring without any problems or clinical signs of impaired welfare, even after multiple lactations. Our results indicate that high amounts of biologically active hIGF-I can be produced in the mammary glands of alpha S1-cas-hIGF-I transgenic rabbits. Local production of hIGF-I in mammary tissue is associated with increased secretion of IGFBP-2, which may prevent major biological effects by high levels of hIGF-I on the mammary gland.