Acute changes in blood flow in pigs infused with beta-adrenergic agonists

Previous results indicate that clenbuterol decreases carcass adipose tissue accretion when administered to pigs but does not appear to stimulate the adipose tissue beta-adrenergic receptor. Clenbuterol increases plasma free fatty acid concentration when acutely infused in vivo, suggesting an indirect affect. One possible indirect effect is that clenbuterol could change blood flow to adipose tissue. Blood flow was measured with radiolabeled microspheres in tissues from pigs before and after infusion of a beta-adrenergic agonist for 30 min. High probability levels (up to P less than .2) were used to indicate trends due to extreme variability. Infusion of isoproterenol increased heart rate, plasma free fatty acid concentration and blood flow at many adipose tissue sites and at a few skeletal muscle sites. Infusion of isoproterenol decreased blood pressure. Infusion of clenbuterol increased heart rate and tended to increase blood flow to several skin and adipose tissue sites slightly. The results suggest that increased adipose tissue blood flow may contribute to the accelerated release of free fatty acids when clenbuterol is infused acutely in vivo.     

beta-Adrenergic receptor agonists increase apoptosis of adipose tissue in mice

beta-Adrenergic receptor (beta-AR) agonists increase muscle mass and decrease body fat in rodents and livestock. With oral administration, however, the effects of beta1-AR and beta2-AR can be different, depending on the species tested. We tested the effects of clenbuterol, a beta2-AR agonist, and ractopamine, a beta1/beta2-AR agonist, on growth, adiposity and adipose tissue apoptosis in male and female mice by feeding diets containing control, 200 ppm clenbuterol, or 200 or 800 ppm ractopamine. Food intake (FI) was measured daily; body weight (BW) and temperatures (BT) were measured on days 0, 3, 7, 10, 14, 17, and 20. On day 21 mice were sacrificed, body composition was determined using PIXImus densitometry, and muscle and adipose tissues were collected. There were no treatment effects on BT, FI, BW, feed efficiency or body composition. Retroperitoneal (Rp) and epididymal/parametrial (Epi/Par) fat pad masses were reduced in both 800 ppm ractopamine (40+/-3mg and 207+/-20mg, respectively) and clenbuterol (35+/-7 mg and 211+/-22 mg) treated mice compared to control (66+/-8 mg and 319+/-30 mg, P<0.05). Brown adipose tissue (BAT) mass was greater (P<0.05) in clenbuterol treated mice compared to other treatments. Adipose tissue apoptosis (% DNA fragmentation) was increased in Epi/Par fat pads in clenbuterol (5.2+/-1.1%) and 800 ppm ractopamine (4.1+/-0.8%) treated mice compared to control (1.7+/-0.4%, P<0.05). These findings show that WAT apoptosis can be induced by activation of beta-AR in mice, although the mechanism is unknown.     

Sensitivity of lipolysis and lipogenesis to dibutyryl-cAMP and beta-adrenergic agonists in swine adipocytes in vitro

The sensitivities of lipolysis and fatty acid synthesis to dibutyryl-cAMP (dbcAMP), epinephrine, ractopamine and clenbuterol were quantified in vitro using porcine adipocytes. Insulin-stimulated lipogenesis showed a biphasic response to dbcAMP, with increased rates at low concentrations and decreased (55%) rates at higher concentrations of dbcAMP. In the absence of insulin, lipogenesis was inhibited 78% by dbcAMP. In the presence of adenosine deaminase or theophylline, all three beta-adrenergic agonists inhibited basal lipogenesis, but only epinephrine and ractopamine inhibited insulin-stimulated lipogenesis. The relationship between suppressed lipogenesis and enhanced lipolysis in response to dbcAMP and the beta-agonists revealed that 1) basal lipogenesis was more sensitive to inhibition than was the stimulation of lipolysis, 2) sensitivity differences were magnified if adenosine deaminase was present and 3) insulin decreased adipocyte sensitivity to the inhibitory effects of dbcAMP and the beta-adrenergic agonists. These results indicate that the relative sensitivities of lipogenesis and lipolysis to beta-adrenergic stimulation can be modified by adenosine and insulin. Furthermore, adenosine and insulin antagonize beta-adrenergic responses, in part, by cAMP-independent mechanisms.     

Hormonal control of porcine adipose tissue fatty acid release and cyclic AMP concentration

Compared with adipose tissue from other mammals, porcine adipose tissue has stringent specificity for stimulation of lipolysis by analogs of norepinephrine. This study was to ascertain whether the specificity for control was reflected in the concentration of tissue cyclic AMP (cAMP). Adipose tissue slices were incubated and concentrations of tissue cAMP and free fatty acids (FA) released to the medium were measured. It was necessary to include theophylline in the incubation medium to be able to measure changes in cAMP concentration. Fatty acid release and cAMP production were increased most effectively by the beta-adrenergic agonists; isoproterenol, fenoterol, dobutamine and the mixed alpha- + beta-adrenergic agonist, epinephrine. Isoproterenol-stimulated FA and cAMP production both inhibited by the beta-adrenergic antagonist, propranolol. There was no evidence for alpha 2-adrenergic inhibition of lipolysis in porcine adipose tissue because clonidine (alpha 2-adrenergic agonist) did not lower isoproterenol-induced FA or cAMP levels and phentolamine, an alpha-adrenergic antagonist, did not increase epinephrine-stimulated FA release or cAMP generation. These results imply that the stringent specificity observed for stimulation of swine adipose tissue lipolysis resides in the beta-adrenergic receptor coupled to cAMP production.     

Acute effects of beta-adrenergic agonists on porcine adipocyte metabolism in vitro

Backfat was obtained at slaughter from market weight hogs to study the acute effects of clenbuterol (CB), ractopamine (RAC) or epinephrine (EPI), in the presence and absence of theophylline (THEO) or adenosine deaminase (ADA), on rates of lipolysis and fatty acid synthesis in vitro. Only EPI increased lipolytic rate in the absence of THEO or ADA. In the presence of THEO or ADA, RAC and CB were lipolytic, although CB had a lower maximal response. With THEO present, RAC and EPI increased lipolysis with a similar potency and responsiveness. Lipolytic responses from all agonists were prevented by propranolol. Insulin stimulated glucose incorporation into fatty acids 50 to 100%; stimulated rates were not influenced by any agonist, either alone or in the presence of ADA. When THEO was present, EPI and RAC inhibited fatty acid synthesis approximately 50%. Clenbuterol was not inhibitory under any conditions. Results indicate that, under appropriate conditions, beta-adrenergic agents increase lipolysis and decrease lipogenesis in porcine adipocytes. Combined evidence suggests that lipolysis is more sensitive to beta-adrenergic stimulation than is insulin-stimulated lipogenesis. Finally, RAC and CB possess only partial agonist activity relative to EPI, CB being least active.     

Effect of dietary clenbuterol and cimaterol on muscle composition, beta-adrenergic and androgen receptor concentrations in broiler chickens

Illegal dietary supplementation with beta(2)-agonists has been shown to increase protein deposition and decrease fat accretion in domestic animals. In poultry the metabolic and endocrine responses to beta(2)-agonists are not fully elucidated. In this trial the effects of dietary clenbuterol (1 p.p.m.) and cimaterol (1 p.p.m.) on muscle composition and endocrine response of male broiler chickens were studied. Dietary clenbuterol induced a slight, but in general not significant, improvement of zootechnical performances and carcass yields. Chemical composition of muscle was not influenced by dietary treatments, even if a slight improvement of protein content was observed in treated groups. No effects on fatty acid composition of meat were detected. Both clenbuterol and cimaterol treatments caused a downregulation in testicular androgen receptors and in pulmonary, cardiac and central nervous system beta-adrenergic receptors.     

Effect of cimaterol on sheep adipose tissue lipid metabolism

Effects of dietary cimaterol (5 mg/kg) on adipose tissue metabolism of wether lambs were studied. Lipogenesis, lipolysis, fatty acid composition and adipocyte size and number were measured. Cimaterol feeding increased lipogenesis; however, this effect was not statistically significant. Insulin (1,000 microU/ml) stimulated lipogenesis of adipose tissue from control sheep. However, this elevated rate was abolished by in vitro cimaterol. Insulin had no stimulatory effect on lipogenesis in cimaterol-fed sheep. Lipolysis was depressed by cimaterol feeding. However, 10(-4) M cimaterol stimulated lipolysis in the adipose tissue from both control and cimaterol-fed sheep. Insulin inhibited stimulated lipolysis in adipose tissue from control sheep but had no effect on the stimulated lipolysis in cimaterol-fed sheep. Mean adipocyte diameter was smaller (from 74 to 70 microns) and adipocyte size distribution also was changed in the cimaterol-fed sheep. Adipocyte number per gram of tissue was not affected by cimaterol. There was a significant increase in percentage of unsaturated fatty acids in adipose tissue from cimaterol-fed sheep. These results indicate that lipogenic and lipolytic responses to insulin and cimaterol in sheep adipose tissue were altered by cimaterol feeding. The carcass fat content decrease in cimaterol-fed sheep may be attributed to the reduction in adipocyte size.     

Growth and adipose tissue metabolism in young pigs fed cimaterol with adequate or low dietary protein

The beta-adrenergic agonist, cimaterol, was fed to young growing pigs to determine whether the carcass compositional changes observed in finishing pigs fed a beta-adrenergic agonist would be manifest in young animals. Furthermore, because cimaterol increased the deposition of lean mass in finishing pigs, it could have a protein sparing effect in young pigs that are rapidly accreting muscle mass and have a high dietary protein requirement. Pigs were fed cimaterol (at 0, .25 and .50 mg X kg-1 diet) and either an adequate (18%) or restricted (14%) protein diet from about 10 to 60 kg body weight. Pigs that were fed the 14 compared with 18% protein diet grew slower and ate less but had the same gain-to-feed ratio. These pigs also had shorter carcasses, less lean muscle and more fat deposition (assessed by carcass measurements and carcass chemical composition) than pigs that received adequate protein. Plasma protein and albumin concentrations were greater and plasma cholesterol, triglyceride and fatty acid concentrations were lower in pigs fed high compared with low dietary protein. Dietary cimaterol had no effect on any of the growth or carcass variables or on adipose tissue metabolism. When fed the high protein diet, cimaterol-supplemented pigs had smaller livers and stomachs. Dietary cimaterol did not have any major detectable effects on these young pigs, nor was there any evidence for a protein sparing effect.     

Effect of ractopamine on insulin sensitivity and response of isolated rat adipocytes

In vitro effects of the phenethanolamine ractopamine on basal and insulin-stimulated lipid metabolism were determined in adipocytes isolated from epididymal fat pads of Sprague-Dawley rats. Ractopamine appeared to be equipotent to the catecholamine isoproterenol in stimulating basal lipolysis and inhibiting basal lipogenesis, producing maximum effects at 10(-6) M. Addition of a half-maximally stimulating dose of ractopamine (5 x 10(-8) M) to the incubation media decreased insulin sensitivity but not insulin responsiveness of the cells, stimulating lipolysis and inhibiting lipogenesis only in the presence of low media insulin concentrations. This effect was totally reversed by 10 microM/propranolol. Maximally effective concentrations of ractopamine (10(-6) M) significantly decreased both the sensitivity and responsiveness of the isolated adipocytes to insulin. Addition of 10 microM propranolol to the incubation media effectively reversed the lipolytic and anti-lipogenic effects of 10(-6) M ractopamine observed at media insulin concentrations greater than 25 microU/ml, whereas it only partially reduced the ractopamine-induced effects observed at lower insulin concentrations. The results demonstrate 1) that ractopamine has concentration-dependent effects on adipose tissue insulin sensitivity and responsiveness and 2) that these effects may be mediated, in part, through beta-adrenergic receptors.     

Influence of infused beta-adrenergic agonists on porcine blood metabolites and catecholamines

Anesthetized pigs were infused sequentially with increased concentrations of beta-adrenergic agonists. At selected times during infusion, blood pressure, heart rate and plasma concentrations of free fatty acids (FFA), glycerol, glucose, lactate, norepinephrine, epinephrine and dopamine were measured. Azaperone, a drug used to calm the pigs before anesthesia, caused hypotension and bradycardia but did not affect plasma metabolites. Infusion of norepinephrine, epinephrine, isoproterenol or clenbuterol produced changes in plasma metabolites and plasma catecholamines. These changes during norepinephrine infusion were attributed to the infused agonist, whereas those during epinephrine infusion might have resulted to some extent from release of norepinephrine. Plasma isoproterenol was not quantified because it interfered with the assay of epinephrine and dopamine so that it was not possible to distinguish between infused isoproterenol and release of endogenous epinephrine and dopamine. Infusion of clenbuterol caused a small increase in plasma norepinephrine so that some of the increase in plasma FFA, glycerol and lactate during clenbuterol infusion may result from release of endogenous norepinephrine.     

Determination of the affinity of ractopamine and clenbuterol for the beta-adrenoceptor of the porcine adipocyte

Dissociation constants (Kd) of ractopamine and clenbuterol for the swine adipocyte beta-adrenergic receptor were estimated from competition studies with epinephrine for the stimulation of lipolysis. Both compounds competitively inhibited epinephrine-stimulated lipolysis in the absence of adenosine deaminase. Three methods for estimating Kd values were used and similar estimates were obtained with each method. Ractopamine and clenbuterol showed greater affinity for the beta-receptor than did epinephrine and had similar Kd values of 1 to 2 x 10(-7) M. The low capacity of ractopamine and clenbuterol to stimulate lipolysis in vitro does not result from poor coupling to the beta-receptor. Ractopamine and clenbuterol may be considered partial agonists, possessing high affinity for the beta-adrenoceptor but exhibiting a relative ineffectiveness for adenylate cyclase activation.     

Decreased insulin binding to porcine adipocytes in vitro by beta-adrenergic agonists

The present study was conducted to determine the influence of dibutyryl-cAMP (dbcAMP), epinephrine, ractopamine and clenbuterol on insulin binding to porcine adipocytes. Dibutyryl-cAMP decreased insulin binding to swine adipocytes by 40 and 20% at 1.8 and 25.8 ng insulin/ml, respectively. Ractopamine and clenbuterol directly reduced insulin binding at the low insulin concentration and decreased binding at high insulin concentrations in the presence of adenosine deaminase. Scatchard analysis suggested that the reduction of insulin binding was due to a decrease in receptor number. Epinephrine alone did not influence insulin binding. In the presence of theophylline, epinephrine decreased binding at both low and high insulin concentrations; however, ractopamine plus theophylline decreased binding only at the low insulin concentration. Clenbuterol did not affect insulin binding in the presence of theophylline. Propranolol blocked the inhibitory effect of epinephrine on insulin binding. These beta-adrenergic agonists can inhibit insulin binding and, thus, antagonize insulin action in swine adipocytes.     

Adipose tissue, longissimus muscle and anterior pituitary growth and function in clenbuterol-fed heifers

The present study was conducted to determine the effects of feeding clenbuterol on adipose tissue and longissimus muscle growth in heifers. For 50 d, 14 heifers were fed either a sucrose-based, clenbuterol supplement or a placebo in which the clenbuterol had been omitted. The heifers were slaughtered in two groups, based on initial weight. Adipose tissue from several anatomical sites and longissimus muscle (depending on slaughter group) were obtained fresh at slaughter. Changes in carcass characteristics elicited by clenbuterol were similar to those reported by others for steers and sheep. Subcutaneous (sc) and intramuscular (im), but not perirenal, adipocytes were smaller and there were more cells per g tissue in the adipose tissue depots of the clenbuterol-fed heifers. Clenbuterol decreased lipogenic enzyme activities, fatty acid-binding protein activity, basal lipolysis and acetate incorporation into glyceride-fatty acids (P less than .05) in sc adipose tissue, but had no effect (P greater than .05) on lipogenesis or lipolysis in im adipose tissue. Clenbuterol elicited a 20% increase in type II myofiber diameters (P less than .05) but had no effect on type I myofiber diameters. In vitro growth hormone release by perifused anterior pituitaries was not affected significantly by long-term in vivo exposure to clenbuterol. These data indicate that a depression in lipogenesis is the mechanism by which clenbuterol decreases subcutaneous fat accretion in cattle.     

Lipid synthesis and adipocyte growth in adipose tissue from sheep chronically fed a beta-adrenergic agent

The effects of the chronic ingestion of the beta-adrenergic agonist clenbuterol on ovine sc adipose tissue were investigated. Three groups of 10 wether lambs with an average initial weight of 22.7 kg were used as experimental animals. After culling 2 to 3 animals per group, one group of eight sheep was slaughtered (initial). The remaining two groups of sheep (control, n = 7 and clenbuterol-fed, n = 8) were fed either a control, high-energy diet or one containing 2 ppm clenbuterol for 40 to 44 d. At slaughter, sc adipose tissue was obtained from all animals for assays in vitro. Subcutaneous fat accretion observed over time in the control sheep was due primarily to an increase in the number of lipid-filled adipocytes. This phenomenon was not observed in the clenbuterol-fed sheep. The incorporation of acetate into lipid increased in the clenbuterol-fed group relative to the initial group and was numerically greater than the rate observed for the control group. Similar results were observed for lipogenic enzyme activities and fatty acid-binding protein activity. Palmitate esterification in vitro tended to be elevated in the clenbuterol-fed group, relative to the control group, suggesting increased triacylglycerol turnover. The in vitro data indicate that clenbuterol did not decrease sc fat accretion in sheep by inhibiting lipogenesis.     

超高效液相色谱-串联质谱法检测猪尿中20种β-受体激动剂残留

建立了猪尿中吡布特罗、西马特罗、特步他林等20种β-受体激动剂残留检测的超高效液相色谱一串联质谱方法。猪尿样品经酶解后,调节pH值至碱性,用叔丁醇和叔丁基甲醚（6＋4,V／V）萃取,MCX柱净化,以0．1％甲酸乙腈溶液和0．1％甲酸水溶液为流动相进行梯度洗脱。同位素内标法和外标法定量。20种β-受体激动剂在系列浓度范围内呈现良好线性关系,相关系数1．2均大于0．99;20种药物在猪尿中的检测限为0．25μg/L,定量限为0．5μg／L。从0．5、1和5μg／L三个添加浓度检测结果可以看出,20种药物的回收率为75．7％～110．7％,批内批间相对杯准偏差均小于20％。该方法具有简便快捷、灵敏度高、定性准确等特点。     

Effects of ractopamine on adipose tissue metabolism and insulin binding in finishing hogs. Interaction with genotype and slaughter weight

Twenty-four barrows were divided among eight treatments in a 2 x 2 x 2 design to quantify the influence of ractopamine (0 or 20 mg/kg diet) over the final 40 kg of gain on metabolic activity in adipose tissue. Interactions with genotype (Hampshire cross or Landrace cross) and slaughter weight (100 or 127 kg) were investigated also. Backfat was removed at slaughter and rates of lipolysis and fatty acid synthesis (FS), activities of malic enzyme (ME) and fatty acid synthetase (FAS), and insulin binding to adipocytes were assessed. Adipocytes from ractopamine-fed pigs were less sensitive (EC50 increased 90%) and had a lower maximum lipolytic response (40%) to ractopamine stimulation. Rates of basal and insulin-stimulated FS were decreased 40% in ractopamine-fed pigs and were reflected in lower activities of ME (50%) and FAS (15%). Breed and slaughter weight had no consistent influence on the ractopamine response. Landrace-cross pigs had greater insulin binding capacity (30-60%) whether data were expressed on a cell or surface area basis. Ractopamine feeding did not consistently affect insulin binding capacity. Results suggest that ractopamine interacts in vivo with the beta-adrenergic receptor of swine adipocytes, decreasing lipogenic capacity and diminishing responsiveness to beta-adrenergic stimulation.     

多壁碳纳米管分散固相萃取结合LC-MS/MS测定饲料中11种β-受体激动剂

本研究建立了采用多壁碳纳米管(MWCNTs)为吸附剂的分散固相萃取(dSPE)净化、液相色谱串联质谱测定饲料中11种β-受体激动剂(克伦特罗、莱克多巴胺、沙丁胺醇、苯乙醇胺A、氯丙那林、妥布特罗、西马特罗、特布他林、马布特罗、班布特罗和喷布特罗)的方法。饲料样品经甲酸水溶液提取后,调节pH值至10,加入50 mgMWCNTs进行分散固相萃取,被吸附的化合物经1.0%甲酸水甲醇(91)洗脱,洗脱液过滤膜后进行LC-MS/MS分析。采用Acquity UPLC BEH C18色谱柱分离,以0.1%甲酸水溶液和甲醇为流动相进行梯度洗脱,电喷雾正电子(ESI+)模式电离,多反应监测(MRM)模式监测,内标法校准进行定量。结果表明:11种β-受体激动剂在0.1～20μg/L浓度范围内呈良好的线性,线性相关系数均大于0.997,猪配合饲料样品中最低定量限为0.05～0.48μg/kg。猪配合饲料中添加0.5～500μg/kgβ-受体激动剂的回收率在89.6%～112.9%,相对标准偏差RSD均小于15%。     

Insulin binding to mouse adipocytes exposed to clenbuterol and ractopamine in vitro and in vivo

Insulin binding to mouse adipocytes was measured after in vitro (30 min) and in vivo (5 days) exposure to clenbuterol and ractopamine. At 10(-6) M, both agonists decreased insulin binding by 20-30% after a 30 min preincubation at each insulin concentration between 1 and 25 ng/ml. Binding was not decreased if propranolol was present. Scatchard plots suggested that decreased binding was due to a decrease in insulin receptor concentration. Insulin binding was decreased approximately 10% at agonist concentrations as low as 10(-13) M, but binding was not further decreased until concentrations exceeded 10(-9) M. Rate of gain was increased 2-fold by clenbuterol (10 mg/liter of drinking water) and 50% by 500 mg ractopamine/liter, but not by 50 mg ractopamine/liter. Clenbuterol and ractopamine (500 mg/liter) decreased fat pad weight but only clenbuterol increased hind limb muscle mass. Insulin binding following in vivo administration was not influenced by ractopamine at 50 mg/liter, but tended to be increased by clenbuterol and ractopamine at 500 mg/liter. The disparity in results between administering the beta-agonists in vitro or in vivo suggests that counter regulatory factors influenced insulin binding capacity in vivo. Results indicate that ractopamine and clenbuterol can decrease insulin binding to adipocytes but the relevance of this response to decreased fat accretion is not clear.     

超高效液相色谱-串联质谱法检测动物肌肉组织中19种β-受体激动剂残留

建立了猪、牛和羊肌肉组织中吡布特罗、西马特罗、特步他林、齐帕特罗、沙丁胺醇、西布特罗、克伦塞罗、克伦丙罗、羟甲基克伦特罗、氯丙那林、莱克多巴胺、克伦特罗、妥布特罗、福莫特罗、溴布特罗、克伦潘特、班布特罗、马布特罗和马喷特罗等19种β-受体激动剂残留检测的超高效液相色谱-串联质谱方法.猪、牛和羊肌肉组织样品用乙腈和异丙醇（8：2,V/V）提取,加入NaC1、Na2SO4和MgSO4盐析去杂质.待测药物经BEH C18色谱柱分离,以0.1％甲酸乙腈溶液和0.1％甲酸水溶液为流动相进行梯度洗脱.同位素内标法和基质匹配标准溶液外标法定量.19种β-受体激动剂在系列浓度范围内呈现良好线性关系,相关系数r2均大于0.99;19种药物在肌肉组织中的检测限为0.25 μg/kg,定量限为0.5μg/kg.从0.5、1和5μg/kg三个添加浓度检测结果可以看出,19种药物的回收率为73.7％ ～ 114.1％,批内批间相对标准偏差均小于20％.结果表明,该方法简便快捷、灵敏度高、定性准确,适用于该类药物残留的检测.     

Effect of bovine growth hormone on incorporation of [14C]acetate into lipids by co-cultures of bovine mammary, liver, and adipose tissue explants

Incorporation of [14C]acetate into lipids was measured in 24 hr co-cultures of mammary, liver and adipose tissue from Holstein cows at 53, 210 and 318 d of lactation in the presence or absence of bovine growth hormone. Little (less than 1%) of the labeled lipids appeared in the media relative to that incorporated into the tissue. In mammary tissue, incorporation of [14C]acetate was highest into triglycerides (16,298 cpm/mg mammary tissue), followed by phospholipids (1,887 cpm), free fatty acids (1,252 cpm), diglycerides (708 cpm), free cholesterol (360 cpm) and monoglycerides (93 cpm). Bovine growth hormone did not increase incorporation of [14C]acetate when mammary or adipose tissue were incubated separately. However, in the presence of liver and adipose tissue, bovine growth hormone significantly increased the incorporation of [14C]acetate into triglycerides, diglycerides, free fatty acids and free cholesterol by mammary tissue. These results suggest that bovine growth hormone acts on mammary tissue indirectly through liver and adipose tissue to increase lipid synthesis. This mechanism may play a role in the action of bovine growth hormone in vivo to increase milk and milk fat production.