Dairy cows suffer from an intense energy deficit at parturition due to the onset of copious milk synthesis and depressed appetite. Despite this deficit, maternal metabolism is almost completely devoted to the support of mammary metabolism. Evidence from rodents suggests that, during periods of nutritional insufficiency, a reduction in plasma leptin serves to co-ordinate energy metabolism. As an initial step to determine if leptin plays this role in periparturient dairy cows, changes in the plasma concentration of leptin were measured during the period from 35 days before to 56 days after parturition. The plasma concentration of leptin was reduced by 50% after parturition and remained depressed during lactation despite a gradual improvement in energy balance; corresponding changes occurred in the abundance of leptin mRNA in white adipose tissue. To determine whether negative energy balance caused this reduction in circulating leptin, cows were either milked or not milked after parturition. Absence of milk removal eliminated the energy deficit of early lactation, and doubled the plasma concentration of leptin. The plasma concentration of leptin was positively correlated with plasma concentrations of insulin and glucose, and negatively correlated with plasma concentrations of growth hormone and non-esterified fatty acids. In conclusion, the energy deficit of periparturient cows causes a sustained reduction in plasma leptin. This reduction could benefit early lactating dairy cows by promoting a faster increase in feed intake and by diverting energy from non-vital functions such as reproduction.

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of 125 I-labeled bovine leptin was linearly related (R 2 = 0·99) to the log of added bovine or ovine leptin between 0·1 to 2·0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor beta and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Ovaries are absolutely required for development of the mammary parenchyma (PAR) in cattle, reflecting estrogendependent epithelial cell proliferation. However, the estrogen receptor (ER) that mediates the mammary estrogen effects, ERa, is absent in proliferating epithelial cells. In the mouse, this discrepancy is explained in part by the ability of the mammary fat pad (MFP) to synthesize epithelial cell mitogens such as IGF-I in response to estrogen. Consistent with a similar role for the bovine MFP, 30% of its fibroblasts and adipocytes were immunoreactive for ERa in prepubertal dairy heifers. To assess estrogen-dependent gene expression in the MFP, 16 prepubertal dairy heifers were randomly assigned to a 2!2 factorial. The first factor was ovarian status, with heifers undergoing bilateral ovariectomy or left intact at 4$6 months of age. The second factor was applied 30 days after surgery and consisted of injection of estrogen or excipient. After 3 days of injection, heifers were administered an intrajugular bolus of bromodeoxyuridine (BrdU) and slaughtered 2 h later. The estrogen injection, but not ovarian status, caused significant increases in the fraction of epithelial cells labeled with BrdU and produced tissue-specific effects on gene expression. In the PAR, estrogen injection increased IGF-I gene expression by twofold despite reductions of 50% or more in ERa mRNA abundance and the fraction of epithelial cells immunoreactive for ERa. The estrogendependent increase in IGF-I mRNA was greater in the MFP, presumably because estrogen failed to downregulate ERa expression in this mammary compartment. Finally, estrogenresponsiveness of the MFP appears unique among the bovine fat depots as estrogen injection did not induce IGF-I expression in its s.c. counterpart. Our data demonstrate that the bovine MFP is highly responsive to exogenous estrogen, consistent with a role for this tissue compartment in communicating its effects on epithelial cell proliferation.

In trial 1, the effects of dietary energy (102, 131 or 162% of requirement) in the dry period and of sodium bicarbonate (0 or .75% of diet dry matter) in early lactation were assessed with 31 cows in a 3 X 2 factorial arrangement of treatments. Body condition and weight increased linearly with prepartum energy. Dry matter intake and milk yield were similar across treatments through 12 wk postpartum. Sodium bicarbonate increased milk fat content only in the 131% group, an effect apparently related to greater mobilization of fat in that group. In trial 2, energy treatments imposed in late lactation (145 to 55 d prepartum) and in the dry period (55 to 0 d) were 1) cows fed to requirement in both periods, 2) cows overfed in the first and underfed in the second period, 3) cows fed to requirement in the first and overfed in the second period, and 4) cows overfed in both periods. Cows in treatments 1 and 2 (normal) calved in a thinner state than those in 3 or 4 (fat). In the first 12 wk postpartum, intake did not differ, but cows in groups 3 and 4 produced more milk. Sodium bicarbonate imposed factorially postpartum increased milk fat content. Overconsumption of energy prepartum did not impair production when high energy total mixed rations were fed postpartum.