Somatotrophin increases thyroxine-5'-monodeiodinase activity in lactating mammary tissue of the cow.

The effect of administration of bovine somatotrophin (bST) on peripheral conversion of thyroxine (T4) to tri-iodothyronine (T3) was studied in non-pregnant lactating Holstein cows. Six cows were injected daily for 5 days with 40 mg recombinantly derived bST, while six control cows received excipient alone. Blood samples were collected hourly from 08.00 to 19.00 h on a single day the week before treatment and on days 4-5 of treatment. All other tissue samples were obtained at slaughter, 20-23 h after the last injection. Administration of bST increased milk production and caused a 9% increase in hepatic DNA. Consumption of feed did not differ between control and bST-treated cows. Treatment did not alter serum concentrations of T4 or T3, although concentrations of thyroid hormones in the serum increased from 08.00 to 19.00 h. Activity of thyroxine-5'-monodeiodinase (5'-D) in liver and kidney was similarly unaffected. However, activity of 5'-D in mammary tissue increased approximately twofold in response to bST administration. We suggest that an increase in mammary conversion of T4 to the more biologically potent thyroid hormone T3 plays a role in mediating the galactopoietic response of dairy cattle to bST.

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells.

A flow cytometric technique was developed to measure the relative concentration of whey protein and beta-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (alpha-lactalbumin + beta-lactoglobulin) or beta-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more beta-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis.

Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 microgram/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [14C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G0G1 and G2M cells and the percentage of cells in the G0G1 population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G2M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G0G1 and G2M cells compared to control but did not affect the percentage of cells in the G0G1, S, or G2M phases. Previously it was assumed that changes in [14C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.

Bovine mammary explant versus primary cell cultures: effect of bovine somatotropin and insulin-like growth factor-I on DNA content and protein synthesis.

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Comparative study of mammary gland development and differentiation between beef and dairy heifers.

Sixteen Hereford and 16 Holstein heifers were used to study the relationship of milk production potential to mammary development and differentiation. Heifers were slaughtered at 150, 180, and 260 days of first gestation and at 49 days of first lactation. Prolactin binding capacity of mammary tissue was 2.5 fold higher in dairy than beef heifers at day 260 of gestation (27.2 vs 11.0 fmols/mg protein). In both breeds, maximal growth hormone binding in liver coincided with the beginning of the rapid phase of mammary growth at 180 days. Mammary tissue from dairy heifers released more casein and alpha-lactalbumin during in vitro incubations than tissue from beef heifers. No differences were observed between breeds with respect to incorporation of [14C]acetate into lipids. Mass of dairy mammary tissue at 49 days of lactation was 3.3 times greater (16.4 vs 4.9 kg) and produced 5.7 times more milk (20.3 vs 3.5 kg/day) than its beef counterpart. The total DNA content and the RNA/DNA ratio of lactating dairy mammary tissue was approximately twice that of lactating beef mammary tissue. The data suggested that the higher milk production observed in dairy cattle is a result of a greater number of secretory cells and greater activity per cell.

Teat stimulation-induced release of prolactin and basal concentrations of prolactin and growth hormone in pregnant dairy and beef heifers.

The physiological bases for differences in milk production between breeds of cattle selected for beef or milk production are largely unknown. This study was conducted to determine concentrations of prolactin (PRL) and growth hormone (GH) in serum before and after teat stimulation in primiparous Hereford and Holstein heifers. Blood was collected from 6 beef and 9 dairy heifers at 115, 175, 230 and 250 d of gestation. Sampling times were -15, -10, -5, 0, 2, 4, 6, 8, 10, 12, 15, 20, 25 and 30 min relative to test stimulation. Mean areas under PRL response curves for beef and dairy heifers at 115, 175, 230 and 250 d of gestation were 427, 447, 556, 273 and 243, 189, 167, 343 ng/ml/30 min, respectively. Calculations of area (but not basal levels) excluded instances when no PRL response to test stimulation was obtained (22%). Neither stage of gestation nor breed affected PRL response. Basal PRL did not differ between breeds and was 1.8, 2.6, 2.4 and 9.2 ng/ml at 115, 175, 230 and 250 d of gestation. GH did not differ between breeds and was 6.6, 6.2, 5.5 and 7.4 ng/ml at 115, 175, 230 and 250 d. No difference between breeds was apparent with regard to PRL or GH secretion during first gestation.

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.

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.

Prolactin and growth hormone binding in mammary and liver tissue of lactating cows.

A radioligand/receptor binding assay was developed using homologous hormones to distinguish between bovine growth hormone (bGH) and bovine prolactin (bPRL) receptors in liver and mammary tissue of lactating cows. Mammary and liver tissues were homogenized in 0.3 M sucrose and centrifuged at 100,000 x g over a 1.3 M sucrose density gradient. Membranes from the 0.3 - 1.3 M sucrose interface were incubated with 1 ng of iodinated bGH or bPRL for 20 h at 22 degrees C in the presence of increasing concentrations of native bGH or bPRL. High affinity receptor binding sites were found for bPRL in liver and mammary tissue membranes (Ka = 3.2 and 1.3 x 10(8) l/mol with 34 and 63 fmol receptors/mg liver and mammary membrane protein, respectively) and for bGH only in liver tissue (Ka = 1.8 x 10(9) l/mol, 18 fmol receptors/mg membrane protein). Receptor number estimates were 3 and 11 times higher in mammary and liver tissue using a heterologous hGH system indicating that heterologous systems may overestimate the number of receptors in bovine tissue. The absence of demonstratable bGH receptors in lactating bovine mammary tissue supports in vitro results of others with isolated mammary tissue indicating that the positive effect of bGH on milk production in intact cows is via an indirect mechanism.

Stocking density effect on weight gain of yearling Holstein heifers in freestall and loose housing.

Stocking densities of replacement yearling Holstein heifers were studied under freestall conditions (1.0, .71, and .63 freestall/head) and loose housing conditions (4.5, 3.2, and 2.8 m2/head under roof) in two 180-day growth trials. Heifers gained .69, .69, and .66 kg/day under freestall conditions and .63, .56, and .71 kg/day under loose housing conditions for the stocking densities. Stocking densities of replacement herd heifers may be increased to .63 freestall/head under freestall conditions or to 2.8 m2/head under loose housing conditions without expanding existing facilities.

Characterization of lactogenic hormone binding to membranes from ovine and bovine mammary gland and liver.

Binding of radiolabeled human growth hormone, bovine prolactin, and ovine prolactin to membranes prepared from ovine and bovine mammary gland and liver was studied. Of these lactogenic hormones, human growth hormone exhibited the greatest total and specific binding capacity to either liver or mammary membranes. Characterization of binding assay conditions of human growth hormone indicated: that divalent ions (calcium or magnesium) were required for maximal binding, that binding was time dependent and saturable, that specific binding was proportional to the quantity of membrane protein assayed, and that bound radiolabeled human growth hormone was displaced similarly with either nonradiolabeled bovine prolactin or ovine prolactin. Interpretation of computer analysis of Scatchard plots derived from displacement curves indicated heterogeneous binding sites in liver and mammary membranes. Mean apparent dissociation constants of the high affinity binding sites ranged from 2.7 to 5.4 X 10(-9) M in mammary and liver membranes, respectively. Compared with mammary membranes from nonlactating ewes, specific binding of human growth hormone was increased 50% on day 100, 190% on day 130 of gestation, and 296% on day 60 of lactation. We conclude that radiolabeled human growth hormone can be used as a probe to measure lactogenic hormone binding sites in liver and mammary membranes from cows and sheep.

Lipid synthesis by co-cultures of mammary, liver, and adipose tissue explants from sometribove (recombinant methionyl bovine somatotropin)-treated dairy cows.

Bovine somatotropin was given to six lactating (230 day) cows (40 mg/day X 5-days) and excipient was given to six control cows. Mammary, liver, and adipose explants from somatotrophin and control cows were co-cultured at 37 degrees C for 24 h with 0.5 microCi [14C]acetate/ml media with or without 0.5 micrograms/ml somatotrophin. Tissue lipids were extracted with chloroform/methanol and separated by thin layer chromatography. In vivo somatotrophin increased milk production 2.4 kg/day compared to a 0.9 kg/day decrease by controls. Mammary tissue from somatotrophin cows incorporated more [14C]acetate into total lipids (4417 vs. 3016 dpm/mg tissue) than controls. Adding somatotrophin to explant cultures from somatotrophin cows further increased incorporation into total lipids (4839 vs. 3994 dpm/mg tissue). In contrast, adipose tissue from somatotrophin cows incorporated less [14C]acetate into total lipids than controls (1524 vs. 2581 dpm/mg tissue). Serum IGF-I concentration correlated well (r = 0.69) with milk output differences between Days 1 and 5 of treatment. Media IGF-I concentration correlated well (r = 0.61) with the difference in total lipid synthesis between the in vitro control and somatotrophin groups. Results support the concept that somatotrophin increases milk production by partitioning nutrients away from adipose toward mammary tissue.

Performance of calves fed fermented mastitic milk, colostrum, and fresh whole milk.

Weight gains, milk intake, and health of calves fed fermented mastitic milk from cows treated and not treated with antibiotics were compared with those of calves fed fermented colostrum or fresh normal milk at two intakes. Calves fed fermented mastitic milk from cows treated with antibiotics, not treated with antibiotics, fermented colostrum (diluted 1:1 with water), and fresh normal milk gained .13, .14, .13, and .10 kg/day in trial 1 fed at 8% of body weight daily, and .09, .11, .18, and .13 dg/day in trial 2 fed at 10% of body weight daily from birth through 30 days of age. All calves were housed in individual pens during milk feeding. Feeding milk at 10% in trial 2 did not improve gains over those in trial 1. Incidence of health disorders and mastitis in first lactation of cows fed fermented mastitic milk as calves was not different from those of cows fed fresh normal milk or fermented colostrum. Fermented mastitic milk appears to be an economical and safe feed for rearing calves when calves are housed individually during milk feeding.

Effect of ratio of corn silage to grass-legume silage with high concentrate during dry period on milk production and health of dairy cows.

Four corn silage: gross legume silage: concentrate totally mixed diets 1) 75:25:0; 2) 48.75:16.25:35; 3) 50:50:0; and 4) 32.5:32.5:5.35 were fed to Holsteins in second and third lactation for two complete lactations. Cows fed diets 1 or 2 during dry period were fed diet 2 during lactation. Diets did not affect length of dry period or lactation, calf weight, milk protein percent, or milk fat percent. Feeding concentrate during dry period increased gain from .63 to 1.11 kg/day. Feeding higher grass-legume silage diets 3 and 4 increased gain during lactation from .25 to .35 kg/day. On a mature equivalent basis, cows fed high corn silage diets 1 and 2 produced more milk (7,105 versus 6,663 kg), protein (236 versus 216 kg), fat (267 versus 245 kg), solids-not-fat (612 versus 564 kg), and a higher solids-not-fat percent (8.62 versus 8.50%) than diets 3 and 4. Diets did not alter health or reproduction. Production and weight gains favored feeding diet 1 during dry period and diet 2 during lactation.

Adrenal response during periparturient period to adrenocorticotropin in dairy cattle fed corn silage and grass legume silage.

Glucocorticoid response to exogenous adrenocorticotropin was used as an indicator of stress in cows fed different diets ad libitum during the dry period and throughout lactation. Twenty-three Holstein cows were administered adrenocorticotropin at 2 d after initiation of the dry period and at 2 d and 35 d postpartum, and plasma glucocorticoid response was evaluated. Cows were fed diets using only corn silage as forage or mixed forage with or without supplemental concentrate. Mean basal plasma glucocorticoid concentrations were 6.1 ng/ml on d 2 after dry off, 4.7 ng/ml on d 2 postpartum, and 6.7 ng/ml on d 35. Cows on the 32.5:32.5:35 corn silage:grass-legume silage:concentrate diet had the lowest basal glucocorticoid concentrations on d 2 postpartum but highest concentrations on d 35 of lactation. Mean glucocorticoid response was 33.2 ng/ml on d 2 of lactation compared with 50.6 ng/ml on d 35 and 48.5 ng/ml plasma on d 2 following drying off. Diet interactions suggest that feed components may be involved with ability to tolerate stresses that occur during lactation and the dry period and thus may lead to altered adrenal function.

Ornithine-delta-aminotransferase in lactating bovine mammary glands.

The occurrence and subcellular distribution of ornithine-delta-aminotransferase have been studied in lactating bovine mammary glands. The enzyme is localized in the mitochondria and has a unique thermal reaction profile that distinguishes it from putative liver and kidney isozymes. The enzyme concentration in the gland correlates well with a role in the conversion of ornithine into the proline precursor, L-delta 1-pyrroline-5-carboxylate. However, an unusually high Michaelis constant for the mitochondrial enzyme (8.4 mM) raises the question of enzyme efficiency in vivo such that this pathway needs to be considered in estimating barriers to protein secretion into milk.

Effect of lactational intensity on extrathyroidal 5'-deiodinase activity in rats.

Concentrations of thyroxine and triiodothyronine in serum and 5'-monodeiodination activity in liver and kidney were studied in lactating Sprague-Dawley rats with different litter sizes. Litter sizes were adjusted at birth to 0 (postpartum nonlactating group), 4, 8, 12, and 16 pups per lactating rat. Serum and tissue samples were collected from lactating rats and pups on d 12 of lactation and from 6 nulliparous females. Nulliparous and postpartum nonlactating rats did not differ in serum thyroxine, triiodothyronine concentrations, or in 5'-deiodinase activity in liver and kidney. As litter size increased, maternal serum thyroxine, triiodothyronine concentrations, and 5'-deiodinase activity in liver and kidney decreased. Growth rate of pups was inversely correlated with litter size and positively correlated with concentration of serum thyroxine and liver 5'-deiodinase in mothers. In pups, serum triiodothyronine concentrations decreased as litter size increased, but serum thyroxine concentrations were not affected. Results suggest a relationship between the hypothyroid status of lactating rats and suckling intensity. The thyroid status of the dam may influence thyroid status and growth of offspring.