Groups of lactating cows and heifers were milked four times daily in two diagonally opposed glands for 4 weeks, and the effects on milk yield studied relative to twice-daily milked glands as controls. Mammary enzyme activities, in vitro synthesis rates of milk constituents and histological scoring were determined in mammary biopsy samples obtained at the end of this period. These were used for assessment of mammary function. Frequent milking increased milk yield only in the treated glands, the contralateral control glands continuing to decline in yield at 2 %/week. There was no significant difference in response between cows and heifers; the mean increase in yield was 10-4%. The rate of decline in milk yield tended to decrease with frequent milking, to ~ 1 %/week. Consequently the yield of the treated glands continued to be elevated above that of the controls for some time after reversion to overall twice daily milking. Milk protein content was increased slightly by frequent milking. Mammary enzyme activities were ~ 18% higher in the treated glands than in the controls. Synthesis rates of lactose, casein and total protein were unaffected by milking frequency, but were all lower in the gland selected for the second biopsy, reflecting the reduction in milk yield caused by the first biopsy. DNA synthesis was increased by milking frequency, as were the size and number of epithelial cells in histological sections.

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of 125I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0.55 nmol/l), less effectively by IGF-II (ED50 8.8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135,000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor alpha-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110-120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (Kd) 2.73 +/- 0.31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0.02) higher (Kd 0.77 +/- 0.06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 +/- 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570 +/- 52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.

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