Developmental plasticity in thyroid function primed by maternal hyperleptinemia in early lactation: a time-course study in rats

Pups whose mothers were leptin-treated during the last 3 days of lactation have thyroid dysfunction at adulthood. However, there was no report about leptin treatment in the first days of life or about its action on thyroid function during development. Here, we evaluated the effects of maternal leptin treatment on the first 10 days of lactation upon thyroid function of the offspring at 21, 30, and 180 days old. At birth, lactating Wistar rats were divided into: Leptin (Lep) - leptin-treated (8 μg/100 g of body weight, s.c.) for the first 10 days of lactation and Control (C, saline-treated). Mothers were killed at the end of lactation and their offspring at 21, 30, and 180 days old. Triiodothyronine (T3), thyroxine (T4), thyrotropin (TSH), and leptin levels in serum and milk were measured. Liver mitochondrial glycerolphosphate dehydrogenase (mGPD) activity was determined. Significant differences had p<0.05. At the end of lactation, Lep mothers had higher milk T3 (+ 30%), while their offspring had higher serum T3 (+ 20%) and TSH (+ 84%). At 30 days-old, Lep offspring showed lower TSH ( - 48%), T3 ( - 20%), and mGPDm ( - 42%). At 180 days-old, Lep group presented hyperleptinemia (1.4-fold increase), higher serum T3 (+ 22%), and lower mGPD activity ( - 57%). Maternal hyperleptinemia on lactation causes hypothyroidism in the pups at 30 days, which may program for higher serum T3 at adulthood. In conclusion, maternal hyperleptinemia during lactation, that is common in obese mothers, may have an impact in future disease development, such as thyroid dysfunction.     

Effects of maternal leptin treatment during lactation on the body weight and leptin resistance of adult offspring

We investigate whether leptin treatment to lactating rats affects food intake, body weight and leptin serum concentration and its anorectic effect on their adult offspring. Lactating rats were divided into 2 groups: Lep-single injected with recombinant rat leptin (8 microg/100 g of body weight, daily for the last 3 consecutive days of lactation) and control group (C) that received the same volume of saline. After weaning all pups had free access to the control diet, their body weight and food intake were monitored at each 4 days until 180 days of age, when they were tested for its food intake and response to either leptin (0.5 mg/kg body wt, ip) or saline vehicle. The offspring of the leptin-treated dams gained more weight and had higher food intake from day 37 onward (p<0.05), higher amount of retroperitoneal white adipose tissue (RPWAT) (37%, p<0.05) and higher leptin serum concentration (40%, p<0.05) at 180 days of age compared to control group. The food intake at 2, 4, 6 and 24 h was unaffected after acute injection of leptin in these animals, suggesting resistance to the anorectic effect of leptin. The maternal leptin treatment during lactation makes their adult offspring more susceptible to overweight with resistance to the anorectic effect of leptin.     

Leptin treatment during lactation increases transfer of iodine through the milk

We have previously shown that protein restriction during lactation is associated with changes in iodine secretion into the milk and that a pup's serum leptin concentration was increased at the end of lactation. So, here we evaluate whether leptin treatment during lactation affects iodine transfer through the milk to the pups. Lactating rats were divided into two groups: the leptin (Lep) group, single injected with recombinant rat leptin (8 microg/100g of body weight, daily for 3 consecutive days), and the control (C) group that received the same volume of saline. We studied iodine transfer to the pups through the milk on Days 4, 12 and 21 of lactation. In those days, the dams were separated from their pups for 4 h. Then, the mothers received an injection of 131I (2.22x10(4) Bq ip) and the pups were allowed to nurse for 2 h. The animals were sacrificed 2 h later. Leptin, total serum T3 and total serum T4 concentrations were higher (P<.05) in pups of Lep mothers only on Day 4, suggesting a higher transfer of leptin through the milk at this period, probably with a direct stimulatory effect on thyroid hormone secretion. In other periods, however, even without a detectable increase in a pup's serum leptin concentration, maternal leptin administration increased the pup's thyroid iodine uptake (Day 12, 39%; Day 21, 34%), probably caused by a higher transfer of iodine through the milk, since they had a higher gastric content of 131I on Days 12 (31%) and 21 (128%).     

Temporal evaluation of the thyroid function of rats programmed by leptin treatment on the neonatal period.

Hormones and malnutrition can imprint several changes in the beginning of life that programs homeostatic changes in the adulthood. We analyzed the thyroid function in 21, 30, 60 and 150 days old animals that were injected with leptin on the first 10 days of life, to determine whether this corresponds to a critical period for the establishment of the hormonal imprinting in the programming of the thyroid function. Pups were divided, within 24 hours of birth, into two groups: Lep group, which was injected once daily with 8 microg/100 g B.W. of recombinant mouse leptin for the first 10 days of lactation, and C-control group that received the same volume of saline. Lep group had higher leptin concentration at days 30 (+6 x , p<0.001) and 150 (+108%, p<0.05) than the controls. These animals had lower serum TT4 (-13%; p<0.05) and TT3 (-17.3%; p<0.002) at 30 days and higher serum TT4 and FT4 concentrations at 150 days (+17.5% and +10%, p<0.05 %, respectively, p<0.05) with lower serum TSH concentrations at 60 (-38.5%, p<0.05) and 150 days (-46%, p<0.05). These animals had also lower hepatic mitochondrial alpha-glycerol-3-phosphate dehydrogenase (mGPDH) activity at 21 (-22.5%; p<0.05), 30 (-50.4%; p<0.05) and 150 days (-40%; p<0.05) than the controls. These data show that the leptin injection in the beginning of lactation cause a hypothyroidism on the offspring as soon as 30 days of age and this alteration may be the imprinted factor for the programming of a higher thyroid function at the adulthood.     

Prolactin inhibition in lactating rats changes leptin transfer through the milk.

Malnutrition during lactation reduces milk production and changes pup's leptin serum levels. To test prolactin role in this nutritional state, we evaluated whether prolactin suppression during lactation changes serum leptin in dams, its transfer through the milk, and pup's serum leptin. Lactating rats were treated with bromocryptine (1 mg/twice a day, s.c.) or saline three days before sacrifice (days 2-4 or days 19-21). Food intake and body weight were measured until sacrifice (4th and 21st day). Serum prolactin and leptin were determined by radioimmunoassay. Bromocryptine injected dams had lower serum prolactin and milk production as expected. The mothers presented lower food ingestion (day 21: -25%), lower body weight (day 4: -12%; day 21: -10%), higher serum leptin (day 4: +68%), lower milk leptin on the 4th day (11 times) and higher (8 times) on the 21st day. The offspring of bromocryptine-treated mothers presented lower body weight in both periods of lactation and lower serum leptin on the 4th day (-40%) and higher on the 21st day (+37%) of lactation. We suggest that prolactin, through its effect on leptin secretion into the milk, may play an important role in signalizing maternal nutritional status to the pups.     

Leptin injection during lactation alters thyroid function in adult rats.

The aim of this study was to evaluate the effects of hyperleptinemia during the first ten days of life on thyroid function in adulthood. After birth, pups were separated into two groups: L8 - receiving daily injections of recombinant mouse leptin (8 microg/100 g body weight, sc) and control (C) - receiving the same volume of saline. Both groups were treated for the first 10 days of lactation. The animals were sacrificed at 150 days of age, and the blood was collected for leptin, TSH, total triiodothyronine (TT 3 ) and total thyroxin (TT 4 ) serum concentration determinations by radioimmunoassay. The thyroid gland was excised to determine thyroid iodine uptake. Leptin, TT 3 and TT 4 serum concentrations in L8 group were significantly (108 %, 47 % and 32 %; p < 0.05) higher than that of controls. There was no significant difference between the groups related to thyroid iodine uptake and TSH serum concentration. These data suggest that the first half of lactation period is important in determining thyroid function in adulthood, and that it can be programmed by serum leptin concentration.     

Neonatal low-protein diet changes deiodinase activities and pituitary TSH response to TRH in adult rats

Protein malnutrition during neonatal programs for a lower body weight and hyperthyroidism in the adult offspring were analyzed. Liver deiodinase is increased in such animals, contributing to the high serum triiodothyronine (T3) levels. The level of deiodinase activities in other tissues is unknown. We analyzed the effect of maternal protein restriction during lactation on thyroid, skeletal muscle, and pituitary deiodinase activities in the adult offspring. For pituitary evaluation, we studied the in vitro, thyrotropin-releasing hormone (TRH)-stimulated thyroid-stimulating hormone (TSH) secretion. Lactating Wistar rats and their pups were divided into a control (C) group, fed a normal diet (23% protein), and a protein-restricted (PR) group, fed a diet containing 8% protein. At weaning, pups in both groups were fed a normal diet until 180 days old. The pituitary gland was incubated before and after TRH stimulation, and released TSH was measured by radioimmunoassay. Deiodinase activities (D1 and D2) were determined by release of (125)I from [(125)I]reverse triiodothyronine (rT3). Maternal protein malnutrition during lactation programs the adult offspring for lower muscle D2 (-43%, P<0.05) and higher muscle D1 (+83%, P<0.05) activities without changes in thyroidal deiodinase activities, higher pituitary D2 activity (1.5 times, P<0.05), and lower TSH response to in vitro TRH (-56%, P<0.05). The evaluations showed that the lower in vivo TSH detected in adult PR hyperthyroid offspring, programmed by neonatal undernutrition, may be caused by an increment of pituitary deiodination. As described for liver, higher skeletal muscle D1 activity suggests a hyperthyroid status. Our data broaden the knowledge about the adaptive changes to malnutrition during lactation and reinforce the concept of neonatal programming of the thyroid function.     

Chronic leptin treatment inhibits liver mitochondrial alpha-glycerol-beta-phosphate dehydrogenase in euthyroid rats.

Leptin modulates the hypothalamus-pituitary-thyroid axis and peripheral metabolism of thyroid hormones (THs). We have studied the effect of acute and chronic leptin treatment upon liver mitochondrial glycerol phosphate dehydrogenase activity (mGPD), whose expression and activity are TH dependent. We performed 2 experiments: 1) acute leptin treatment - LepA: adult rats received a single leptin injection (8 microg/100 g BW); 2) chronic leptin treatment - LepC: adult rats received leptin (8 microg/100 g BW) daily, for 6 days. In both experiments, control groups were saline-treated. All rats were sacrificed 2 hours after the last dose. Liver mGPD activity was determined by colorimetric method. Liver D1 activity was measured by the release of (125)I from (125)I-rT3. Serum hormones were measured by RIA. LepA rats showed higher serum thyroid stimulating hormone (TSH) (+ 64%, p<0.05), free T4 (+ 34%, p<0.05), free T3 (+ 64%, p<0.05), and liver D1 activity (+ 85%, p<0.05), but no change in mGPD activity. Since THs increase mGPD activity, the unchanged level in the acute experiment is suggestive of an inhibitory role of leptin. LepC rats presented lower mGPD activity (-1.7-fold, p<0.05) and higher liver D1 activity (+ 32%, p<0.05), but no alteration in serum TSH and free THs. Our results show that leptin downregulates mGPD activity, mainly when hyperleptinemia is chronic.     

Leptin and prolactin, but not corticosterone, modulate body weight and thyroid function in protein-malnourished lactating rats.

To understand the role of hormonal changes in the lower food ingestion and body weight in protein-restricted lactating rats as well as the higher serum T (3), higher deiodination, iodide and T (3) milk transfer, we measured maternal serum prolactin, leptin, TSH and corticosterone, which are hormones that could influence those parameters. After birth, dams were separated into: control-fed with a 23 % protein diet (n = 12) and PR (protein-restricted)-fed with an 8 % protein diet (n = 12). At the 4 (th) and 21 (st) day of lactation, half of the animals in each group were sacrificed. PR dams presented hyperleptinemia (day 4: + 20 %; day 21: + 19 %; p < 0.05) and hypoprolactinemia (day 4: - 85 %; day 21: - 92 %; p < 0.05), which could help explain the lower food consumption and body weight in lactating PR rats since leptin is anorexigenic and prolactin is orexigenic. Also, this hyperleptinemia could contribute for the increase in serum T (3) of PR dams, since leptin stimulates T (3) production, especially acting on deiodinases. Serum corticosterone was not different between PR and C groups, and TSH was lower only at the end of lactation. Thus, we suggest that both leptin and prolactin could play an important role in the body weight and thyroid hormone changes observed in protein-malnourished lactating rats.     

Developmental plasticity in adrenal function and leptin production primed by nicotine exposure during lactation: gender differences in rats

Neonate male rats whose mothers were nicotine-treated during lactation have higher adiposity, hyperleptinemia, and adrenal dysfunction. At adulthood, they still present higher adiposity and hyperleptinemia, but there was no report about their adrenal function. Also, there was no report of this developmental plasticity on females. Here, we evaluated the adrenal function and leptin content in adipocytes and muscle of male and female adult offspring whose mothers were nicotine-treated during lactation. On the 2nd postnatal day (PN2), dams were subcutaneously implanted with osmotic minipumps releasing nicotine (NIC-6?mg/kg/day) or saline for 14 days (12 litters/group and 2 rats/litter). Male and female offspring were killed on PN180. Significant data were p<0.05. Male NIC offspring presented higher adrenal catecholamine content (+?89%) and TH expression (+?38%), lower "in vitro" catecholamine release (-?19%), and higher adrenergic β3 receptor (ADRB3, +?59%) content in visceral adipose tissue (VAT). Serum corticosterone was higher (+?77%) in male NIC group, coherent with the increase of both CRH and ACTH immunostaining in hypothalamus and pituitary, respectively. Leptin content was higher in VAT (+?23%), which may justify the observed hyperleptinemia. Female NIC offspring presented lower ADRB3 content in VAT (-?39%) and lower leptin content in subcutaneous adipose tissue (SAT) (-?46%), but higher leptin content in soleus muscle (+?22%), although leptinemia was normal. We evidenced a sex dimorphism in the model of maternal nicotine exposure during lactation. The adrenal function in adult offspring was primed only in male offspring while the female offspring displayed relevant alterations in leptin content on muscle and adipocytes.     

Effects of leptin supplementation to lactating Brandt’s voles (<Emphasis Type="Italic">Lasiopodomys</Emphasis><Emphasis Type="Italic">brandtii</Emphasis>) on the developmental responses of their offspring to a high-fat diet

Maternal serum leptin concentrations have been suggested as a key factor in programming growth patterns and protecting against adult metabolic disease in human offspring. However, the role of maternal leptin in the development of wild rodent offspring is not clear. We tested the hypothesis that maternal hyperleptinemia in lactating Brandt’s voles (Lasiopodomys brandtii) can protect their offspring from the risks of high-fat-diet-induced-obesity and insulin resistance. Lactating voles were supplemented with murine leptin (0.64 μg g⁻¹ day⁻¹) or phosphate-buffered saline (control) on days10–17 of lactation (peak lactation). At 12 weeks of age, the female and male offspring of the two maternal groups were randomly assigned to two groups each and fed either a high-fat diet (41% of gross energy as fat) or a control diet (14% of gross energy as fat) until the age of 23 weeks. Body mass, food intake, glucose tolerance and resting metabolic rate were determined in the four offspring groups. After animals were sacrificed, organ masses and adipose tissue distribution, and serum leptin and insulin concentrations were measured. Offspring of leptin-treated mothers showed no significant differences in body mass, energy intake or energy expenditure, body composition, glucose tolerance or serum leptin and insulin concentrations from offspring of control mothers. The high-fat diet induced increases in body mass (by 23% in female and 17% in male offspring) and reduced glucose tolerance in both female and male offspring, indicative of the emergence of insulin resistance, even though digestible energy intake of the male offspring decreased on the high-fat diet. These results indicate that maternal hyperleptinemia during peak lactation in Brandt’s voles did not protect against diet-induced obesity or glucose intolerance in their offspring.     

Leptin-programmed rats respond to cold exposure changing hypothalamic leptin receptor and thyroid function differently from cold-exposed controls

We showed that neonatal leptin treatment programmes for hyperleptinemia and central leptin resistance both at 30days-old and adulthood, while programmes for lower serum T3 at 30days-old, but higher thyroid hormones (TH) at adulthood. As in these animals, acute cold at 30days-old normalized leptinemia and restored the expression of hypothalamic leptin receptor (OBR), here we evaluate the effect of cold exposure on the thyroid function and OBR in adult rats programmed by neonatal hyperleptinemia. Pups were divided into 2 groups: Lep-injected with leptin (8μg/100g/BW, sc) for the first 10days of lactation, and C-injected with saline. At 150days, both groups were subdivided into: LepC and CC, which were exposed to 8°C for 12h. Serum leptin, TH, TSH, liver type I and brown adipose tissue (BAT) type II deiodinases (D1 and D2) activities, liver mitochondrial alpha-glycerol-3-phosphate dehydrogenase (mGPD) activity and adrenal catecholamine content were measured. Hypothalamic and thyroid OBR protein contents were evaluated. Differences were significant when p<0.05. Lep group had hyperleptinemia (+19%), higher T4 (+20%) and T3 (+30%) with lower TSH (-55%), higher liver D1 (1.4 fold-increase), lower BAT D2 (-44%) and liver mGPD activities (-55%), higher adrenal catecholamines (+44%), lower hypothalamic OBR (-51%) and normal thyroid OBR. Cold exposure normalized leptinemia, D1, mGPD, catecholamine and hypothalamic OBR. However, cold exposure further increased TH and decreased D2. Thus, cold exposure normalizes most of the changes programmed by neonatal hyperleptinemia, at the expense of worsening the hyperthyroidism and BAT thermogenesis.     

Leptin serum concentration,food intake and body weight in rats whose mothers were exposed to malnutrition during lactation

We had shown that adult animals, whose mothers were submitted to protein or energy restriction during lactation, differ from controls in their body weight and thyroid function. The aim of this study was to evaluate, from birth through six months of age, leptin serum concentration, body weight and food intake in animals whose mothers received protein or energy restricted-diet during lactation as follows: control (C)-23% protein; protein-restricted (PR)-8% protein; energy-restricted (ER)-23% protein, in restricted quantity, according to the mean ingestion of the PR group. After weaning (day 21) all pups had free access the control diet. Body weight of pups from PR mothers were always lower than those from controls (p < 0.05), while body weight of pups from ER mothers surpassed that of the C group significantly at 140 days of age. The food intake was lower in both offspring from PR and ER mothers, normalizing on the 32th day in pups from ER mothers and on the 52th day in pups from PR mothers. Leptin serum concentration in both offspring from PR and ER mothers were significantly decreased on the 12th day (p < 0.05) and increased on the 21st day (p < 0.05) compared to control. After weaning there was no differences among the groups. It is possible that changes in leptin concentration during lactation in the offspring of malnourished groups could permanently modify the setpoint for body weight control.     

Long-term effects of malnutrition during lactation on the thyroid function of offspring.

Some studies have shown that the mother's nutritional condition may influence offspring's endocrine function through metabolic imprinting. Recently, we showed that the kind of maternal malnutrition during lactation affects adult body weight of the offspring and it is related to milk composition. We studied lactating rats fed an 8 % protein-restricted diet (PR), a control 23 % protein diet (C), and an energy-restricted diet group (ER). After weaning, all animals received a normal diet until they were 180 days of age. At this time, the animals received a single i. p. injection of (131)I and were sacrificed 2 h after the injection. Total triiodothyronine (TT3) and total thyroxin (TT4) serum concentrations were measured by enzyme immunoassay. The PR group had significantly a higher thyroid (131)I uptake, TT3 serum concentration and in TT4 serum concentration, compared to the controls. The ER group had only significantly higher TT3 serum concentration. These results showed that thyroid function regulation in adulthood may depend on maternal nutritional condition during lactation. Probably, PR group had a high thyroid function, whereas the ER group only had an increase in the deiodination of T4. The hyperthyroidism in the PR group could explain the low body weight observed in those animals.     

Maternal prolactin inhibition during lactation is associated to renal dysfunction in their adult rat offspring

The renal function of rats whose mothers had hypoprolactinemia at the end of lactation was evaluated during development. Lactating Wistar rats were treated with bromocriptine (BRO, 1?mg twice a day, s.c.) or saline on days 19, 20, and 21 of lactation, and their male offspring were followed from weaning until 180 days old. 1 rat from each of the 12 litters/group was evaluated at 2 time points (90 and 180 days). Body and kidney weights, sodium, potassium, and creatinine were measured. Values were considered significant when p<0.05. Adult BRO-treated offspring presented higher body weight (+10%), lower relative renal weight at 90 and 180 days (-9.2% and -15.7%, respectively), glomerulosclerosis, and peritubular fibrosis. At 90 and 180 days, creatinine clearance was lower (-32% and -30%, respectively), whereas serum potassium was higher (+19% and +29%, respectively), but there were no changes in serum sodium. At 180 days, higher proteinuria (+36%) and serum creatinine levels (+20%) were detected. Our data suggest that prolactin inhibition during late lactation programs renal function damage in adult offspring that develops gradually, first affecting the creatinine clearance and potassium serum levels with further development of hyperproteinuria and higher serum creatinine, without affecting sodium. Thus, precocious weaning programs some components of the metabolic syndrome, which can be a risk factor for further development of kidney disease.     

Acute cold exposure,leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of (125)I from (125)I-reverse triiodothyronine (rT(3)) under different assay conditions. Rats exposed to 4 degrees C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching approximately 40% at 2 h (P < 0.05) despite increased circulating TSH (P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 microg/100 g body wt sc) induced increased thyroid and liver D1 (P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 microg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.     

Functional state of the thyroid gland during systemic graft vs host reaction

Triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) serum content was measured in mice during systemic "graft-versus-host" reaction (GVHR), using radioimmunoassay. It was demonstrated that on the 3rd day after GVHR induction the levels of these hormones did not differ from the control values. T3 and T4 concentrations and 125I absorption by thyroid gland diminished by day 10. At the same time TSH level remained unchanged. On day 24 after GVHR induction T3 and T4 content was significantly reduced, although TSH concentration exceeded the control value. 125I absorption was enhanced as compared to the value observed on day 10. The data obtained show the vigorous inhibition of thyroid gland function during systemic GVHR.     

Maternal tobacco smoke exposure during lactation inhibits catecholamine production by adrenal medullae in adult rat offspring

Previously, we have shown that maternal smoke exposure during lactation, even when pups are not exposed, affects biochemical profiles in the offspring at weaning, eliciting lower body adiposity, hyperinsulinemia, hypocorticosteronemia and lower adrenal catecholamine content. However, the future impact of tobacco exposure is still unknown. As postnatal nicotine exposure causes short- and long-term effects on pups' biochemistry and endocrine profiles, we have now evaluated some endocrine and metabolic parameters of the adult offspring whose mothers were tobacco exposed during lactation. For this, from day 3 to 21 of lactation, rat dams were divided in: 1) SE group, cigarette smoke-exposed (1.7 mg nicotine/cigarettes for 1 h, 4 times/day, daily), without their pups, and 2) C group, exposed to air, in the same conditions. Offspring were killed at 180-days-old. Body weight and food intake were evaluated. Blood, white adipose tissue, adrenal, and liver were collected. All significant data were p<0.05. The adult SE offspring showed no change in body weight, cumulative food intake, serum hormone profile, serum lipid profile, or triglycerides content in liver. However, in adrenal gland, adult SE offspring showed lower catecholamine content ( - 50%) and lower tyrosine hydroxylase protein expression ( - 56%). Despite the hormonal alterations during lactation, tobacco smoke exposure through breast milk only programmed the adrenal medullary function at adulthood and this dysfunction can have consequence on stress response. Thus, an environment free of smoke during lactation period is essential to improve health outcomes in adult offspring.     

Thyroxine and triiodothyronine in milk of cows, goats, sheep, and guinea pigs

Milk was collected for the first 21 days of lactation twice daily from dairy cows and once daily from goats, sheep, and guinea pigs. Thyroxine (T4) and triiodothyronine (T3) were extracted from 100 microliter of milk using acidified ethanol. T4 and T3 were reconstituted in 100 microliter buffer and measured by radioimmunoassay. Concentrations (ng/ml) of T4 and T3 for milk of cows, goats, sheep, and guinea pigs, respectively, were: 0.97 and 0.94, 1.24 and 0.52, 0.99 and 0.79, and 1.41 and 0.53. T4 concentration for guinea pig milk was significantly higher than for cow and sheep milk, but not for goat milk (P less than 0.05). T3 was found in higher concentration in milk of cows and sheep than in milk of goats and guinea pigs (P less than 0.05). Species differences in conversion of T4 to T3 in mammary gland cells are suggested. Summations of T4 and T3 concentrations in milk indicated no differences among the four species. Regression analyses of changes in milk production, T4 and T3 concentrations, total T4 and T3 in milk per day, and ratios of T4 to T3 revealed variations in patterns. Concentrations of T4 or T3 tended to decrease as lactation progressed over 21 days. Total T3 tended to increase, and the ratio of T4 to T3 tended to decrease. Amounts of T4 and T3 available to offspring from milk were calculated to be minor sources (4 to 7%) of total requirements for maintenance of metabolic functions.