Short communication: Staphylococcus aureus infection modulates expression of drug transporters and inflammatory biomarkers in mouse mammary gland.

Mastitis is the most common disease in dairy herds worldwide and is often caused by Staphylococcus aureus. Little is known about the effect of mastitis on transporters in the mammary gland and the effect on transporter-mediated secretion of drugs into milk. We studied gene expressions of ATP-binding cassette and solute carrier transporters in S. aureus-infected mammary glands of mice. On d 7 of lactation, NMRI mice were inoculated with 1,000 cfu of S. aureus in 2 mammary glands and with a saline vehicle in 2 control glands. Gene expression of the transporters, Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1, and Oct1, and of Csn2, the gene encoding ß-casein, were determined in mammary glands at 72 h after treatment. As biomarkers of the inflammatory response gene, expressions of the cytokines Il6, Tnfα, and the chemokine Cxcl2 were measured. Despite a high individual variation between the 6 animals, some characteristic patterns were evident. The 3 inflammatory biomarkers were upregulated in all animals; Csn2 was downregulated compared with controls in all animals, although not statistically significantly. Both Mrp1 and Oatp1a5 were statistically significantly upregulated and Bcrp was downregulated. Gene expression of Bcrp followed the expression of Csn2 in each of the animals, indicating a possible co-regulation. The findings demonstrate that S. aureus infection has an effect on expression of drug transporters in the mammary gland, which may affect secretion of drugs into milk and efficacy of drug therapy.

Review: Sensor techniques in ruminants: more than fitness trackers.

In this position paper, I shall summarise the current status of sensor technologies in ruminant livestock farming with emphasis on dairy cattle, outline the case for why I believe that sensor technologies could revolutionise global dairy farming in a positive way, describe the significant barriers that exist if that goal is to be achieved and highlight the benefits to animal wellbeing, profitability and sustainability that could result if the technologies are implemented to a significant extent. I shall not provide a comprehensive review of the sensor technology literature since that has been done before, but I intend to provide a sensible amount of background information and data that will allow the reader to obtain a picture not only of today's sensor usage but, more importantly, the possible future direction of dairy animal-oriented sensor technologies, and I shall substantiate my claims and conclusions with relevant literature.

Oxytocin injections in the postpartal period affect mammary tight junctions in sows.

The potential impacts of injecting oxytocin (OXY) to sows in the early postpartum period on the quality of mammary tight junctions, milk composition, and immune status of sows and piglets were studied. Postparturient sows received i.m. injections of either saline (control [CTL]; = 10) or 75 IU of OXY ( = 10). Injections were given twice daily (0800 and 1630 h) starting on d 2 of lactation (i.e., between 12 and 20 h after birth of the last piglet), totaling 4 injections. Milk samples were obtained before the first injection (d 2 morning [AM]), before the second injection (d 2 afternoon [PM]), and on d 4 PM and d 5 PM. Blood samples were obtained from sows before milking on d 2 AM, d 2 PM, and d 5 PM. On d 5 of lactation, a blood sample was obtained from 3 piglets per litter. Circulating concentrations of prolactin, IGF-I, lactose, and IgA in sows did not differ between treatments at any time ( > 0.10), but OXY sows had less IgG than CTL sows ( < 0.01) on d 2 PM before the second OXY injection. There were differences in milk composition on d 2 PM, with OXY sows having more IGF-I ( < 0.01), solids ( < 0.05), protein ( < 0.01), energy ( < 0.05), and IgA ( < 0.01) and a greater Na:K ratio ( < 0.01) than CTL sows. These differences were not seen in the next 2 milk samples, except for protein and IgA that still tended ( < 0.10) to be greater in OXY vs. CTL sows on d 4 PM (for protein) and on d 5 PM (for IgA) after the last injection. Milk lactose content was lower in OXY vs. CTL sows on d 5 PM ( < 0.01). Values for immunoglobulin immunocrit, IgG, IgA, and IGF-I in piglet blood did not differ between treatments ( > 0.10). Injecting OXY to sows in the early postpartum period increased leakiness of the mammary tight junctions, improved composition of early milk, and may potentially affect immune status of neonatal piglets.

Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis.

The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M-SAA3 in mammary tissues was determined using immunocytochemistry. Increases of M-SAA3 and Hp in milk occurred within 12 h of Staphylococcus aureus infusion, with peak concentrations occurring 3 d after infusion of the bacteria. The increase of acute phase proteins in milk (15 h) preceded the increase in serum concentrations of both proteins (24 h). Expression of mRNA for M-SAA3 and Hp increased in both mammary and hepatic tissues 48 h after infusion of the mammary glands. In mammary tissue, the increase of M-SAA3 mRNA was greater than the increase in Hp mRNA expression, whereas in hepatic tissue, the increase in M-SAA3 mRNA was less than that for Hp mRNA. Immunocytochemistry demonstrated that M-SAA3 protein was present within secretory epithelial cells at significantly higher levels in infected mammary glands than in control tissues. These proteins, which have host defense and antibacterial activities, may play a significant role in the early response to invasion of mammary tissues by pathogenic bacteria.

Effects of supplementing pregnant heifers with methionine or melatonin on the anatomy and other characteristics of their lateral hind claws.

Three groups of four primiparous Holstein-Friesian heifers were fed throughout pregnancy either a control diet or that diet supplemented with either 5 to 6 g per day of rumen-protected intestinally available methionine or 25 mg melatonin. They were euthanased three days after calving. The dietary supplements had no effect on the impression hardness or the concentrations of cysteine and methionine in samples of claw horn collected from a range of sites, or on the areas of erosion in the sole and heel. Significant differences were recorded for the hardness of the horn in the order wall >sole >heel. These differences were associated with higher concentrations of cysteine and lower concentrations of methionine in samples of horn from the dorsal wall than in samples from the prebulbar region of the sole. There were no significant differences attributable to the dietary supplements in the soft tissue anatomy of the solear dermis and epidermis.

Suitability of saliva cortisol as a biomarker for hypothalamic-pituitary-adrenal axis activation assessment, effects of feeding actions, and immunostimulatory challenges in dairy cows.

One of the most prominent physiological responses to stressors is the activation of the hypothalamic-pituitary-adrenocortical (HPA) axis, currently assessed by measuring cortisol concentration in blood plasma. To reduce animal discomfort during sampling, which negatively affects stress biomarkers, current research focuses on noninvasive sampling of media other than blood, for example, saliva. The aim of this study was to assess the suitability of saliva cortisol as a biomarker under different physiological and immunological states in dairy cows. Our objectives were to 1) evaluate the relationship between HPA axis activation and saliva cortisol concentration, 2) investigate effects of some feeding action (as influenced by feed and water consumption) on saliva cortisol concentration, and 3) evaluate the time lag between plasma and saliva cortisol during induced inflammatory conditions by intramammary lipopolysaccharide (LPS) and lipoteichoic acid (LTA) injection. During a specific activation of the HPA axis, a positive correlation ( = 0.75, < 0.0001) between saliva and blood cortisol concentrations was observed with increased ( < 0.01) plasma cortisol concentrations following ACTH administration. Saliva and blood samples were taken before, during, and after drinking, feeding, and ruminating. Only a low correlation between saliva and plasma cortisol concentrations ( = 0.03, = 0.83) but no significant effects of the different feeding actions on saliva cortisol were observed. When compared with basal concentrations, cortisol concentrations in plasma significantly increased during inflammatory responses following LPS and LTA injection. Compared with plasma cortisol, changes in saliva cortisol concentrations occurred at a much lower level within a narrow range and did not necessarily follow changes in plasma. In conclusion, the positive correlation between saliva and plasma cortisol concentration in response to ACTH and inflammation suggests the suitability of saliva cortisol measurement for the HPA axis activation assessment. However, changes in saliva cortisol concentration occur within a very narrow range. Furthermore, not only must variation among individual animals be considered but also variation within the same animal. Only with additional knowledge of the concomitant physiological status of the cow it is possible to correctly evaluate saliva and blood cortisol samples.

Regulation of intracellular casein degradation by secreted milk proteins.

Intracellular degradation of newly synthesised casein was measured by a pulse-chase method in freshly prepared goat mammary explants. After incubation in medium containing L-[5-3H]proline, explants were washed and cultured again in unlabelled medium containing 5 mM proline; at intervals up to 24 h the amount of radiolabel incorporated in casein was measured. Tissue was obtained in week 33 of lactation after goats had been milked incompletely in one gland (the test gland) for 24 weeks; the contra-lateral (control) gland was milked normally. In explants from the control gland, casein was not degraded during or after secretion: L-[5-3H]proline incorporated in casein increased to a maximum value which was maintained through the chase period. For four out of five goats, explants from the test gland showed a decrease in total [3H]casein radiolabel at 0-4 h of the chase, indicating that a proportion of casein was degraded during secretion. Intracellular casein degradation was also observed when control gland explants were cultured in chase medium containing a goat whey fraction known to inhibit casein production and milk secretion (Wilde, C.J. et al., (1987) Biochem. J. 242, 285-288). This suggests that the greater volume of residual milk left by incomplete milking reduced secretory efficiency, rendering casein susceptible to intracellular degradation, and that this occurred through the action of a secreted milk constituent, which acts as a chemical feedback inhibitor of milk secretion.

Local control of the mammary gland.

Studies on increasing the frequency of milking in dairy animals have led to the uncovering of the mechanism by which tactical control of the rate of milk secretion is achieved locally within each mammary gland, against a strategic, systemic control by the hormones that maintain all glands in the secretory condition. Experiments in vivo established that the response is local, and were compatible with the hypothesis that milk contains an inhibitor of its own secretion which accumulates during storage within the lumen of the mammary gland and which acts in an autocrine manner on the secretory cells. Isolation of a protein, initially from goats' milk, called FIL (feedback inhibitor of lactation) has enabled, and is enabling, further studies to be done from the whole-animal down to the molecular level. Examples at the whole-animal level are: the effects of immunization against FIL on the rate of secretion; the concentration of FIL and the kinetics of its formation and breakdown; the importance of the internal structure of the mammary gland and the capacities of the alveolar and ductular storage regions in determining feedback inhibition; differences between individuals and species influencing the degree of control exerted by FIL in matching supply of milk to demand by the young. Other local control mechanisms at the onset and cessation of lactation, including mammary distension, are also discussed.

Autocrine regulation of milk secretion.

Mammary development and the rate of milk secretion are regulated by frequency and completeness of milk removal. This regulation occurs through chemical feedback inhibition by a milk constituent. Novel, immunologically related milk proteins able to perform this function have been isolated from caprine, bovine and human milk, based on their ability to inhibit milk constituent synthesis in mammary tissue and cell cultures, and to decrease temporarily milk secretion when added to milk stored in the mammary gland. Inhibition is concentration-dependent, suggesting that milk accumulation and removal is accompanied by cyclical changes in inhibitor accretion and depletion in milk. Feedback inhibition is an autocrine mechanism: the caprine inhibitor, termed FIL (feedback inhibitor of lactation) is synthesized by mammary epithelial cells in primary culture. Inhibition is by reversible blockade of the secretory pathway, an effect which, by down-regulating cell-surface hormone receptors, has longer-term consequences on epithelial cell differentiation. Treatment of goat mammary epithelial cell cultures with caprine FIL initially decreased milk protein secretion and subsequently reduced milk protein messenger RNA abundance. Thus the actions of a single milk constituent can bring about both the effect of milking frequency on milk secretion rate and a sequential modulation of cellular differentiation which acts to sustain the secretory response. Long-term regulation, through changes in galactopoietic hormone receptors, also provides an efficient mechanism for integrating acute intramammary regulation of lactation with strategic endocrine control of mammary tissue development.

Short-term oxytocin treatment increases bovine milk yield by enhancing milk removal without any direct action on mammary metabolism.

The milk yield-enhancing effect of oxytocin administered either before or after milking was examined using a within-animal model. Eight cows in mid-lactation were changed from normal twice daily milking to a split-milking design, whereby half of the udder was milked at 0500 and 1500 h (control half; normal milking times), and the other half at 0800 and 1800 h (test half). This continued for 3 weeks. During the second week, oxytocin was administered as an i.m. injection immediately before the 0800 and 1800 h milkings. The test half was thus milked immediately after oxytocin administration, whilst the control half was milked 3 h before oxytocin. Milk yield decreased slightly on the adoption of split-milking. The decrease did not differ between udder halves and was not, therefore, due to inadequate milk-ejection in the test half compared with the control. During the week of oxytocin treatment, the yield decreased further in the control half but increased in the test half; consequently, the yield from the test half was significantly greater than that from the control half (P < 0.05). The ratio of change in the test half relative to that in the control half was 1.12, significantly different from unity (P = 0.002). Analysis of variance demonstrated a significant (P < 0.001) interaction between udder half and oxytocin treatment, confirming that the effect of oxytocin was restricted to the test half, treated immediately before milking. This supports the established view that oxytocin acts by enhancing the milk-ejection reflex, and refutes a recent claim that the hormone has a direct stimulatory action on mammary metabolism.

Effects of prolactin, progesterone and ovariectomy on metabolic activities and insulin receptors in the mammary gland and adipose tissue during extended lactation in the rat.

Prolactin implants prevented the decline in milk yield and the resumption of oestrous cycles which occurred between days 22 and 28 in untreated lactating rats. Ovariectomy and progesterone implants only partially prevented the decline in milk yield despite preventing the occurrence of oestrous cycles. All three treatments increased total RNA content of the mammary gland compared with controls. In untreated rats there were no changes in mammary DNA content or the number of insulin receptors whereas lipoprotein lipase (LPL) activity decreased significantly during the declining phase of lactation. In contrast, the number of insulin receptors, LPL activity and glucose incorporation into lipid increased in adipose tissue. Prolactin prevented the increase in insulin receptors and lipid synthesis and significantly decreased LPL activity in adipose tissue. Progesterone stimulated LPL activity in the mammary gland and also prevented the increase in lipid synthesis and insulin receptors in adipose tissue but was without effect on LPL activity whereas ovariectomy stimulated LPL activity in the mammary gland but prevented only the increase in the number of insulin receptors in adipose tissue. The results show that raising the serum prolactin concentration can prevent the decline in milk yield during extended lactation and whilst part of this effect may be due to a direct effect on the mammary gland and an indirect effect due to inhibition of oestrous cycles, prolactin may also produce part of its effect on milk synthesis by inhibiting competitive metabolic processes in tissues such as adipose tissue.

Stimulation of milk secretion with inhibition of milk ejection by corticosteroids during extended lactation in the rat.

Milk yield declined significantly between days 22 and 28 of lactation in rats, when lactation was extended by frequent replacement of older litters with younger ones. Corticosterone implants but not cortisol injections or implants prevented this decline. Cortisol, however, appeared to inhibit milk ejection since the mammary glands became engorged with milk and milk yield was improved dramatically by oxytocin injections. In both cases corticosteroid concentrations increased approximately threefold above basal concentrations. Both corticosteroids increased total mammary gland RNA content and lipoprotein lipase (LPL) activity of the mammary gland but were without effect on insulin binding. They also decreased LPL activity, lipogenesis and the number of insulin receptors on adipose tissue. Serum prolactin and insulin concentrations were unaffected by any of the treatments. The results suggest that corticosteroids inhibit milk ejection under certain conditions, may be circulating in lower concentrations, which thereby limit milk production, during prolonged lactation and may improve milk yield during extended lactation in part by suppressing anabolic activity in adipose tissue.

Inhibitory effects of bromocriptine on mammary development and function in lactating mice.

Five experiments were conducted. In the first, the plasma prolactin concentration of lactating mice was significantly reduced 3, 6 and 12 h after a single injection of 0.2 mg bromocriptine at 09.00 h, but recovered to normal levels at 24 h. A second injection at 17.00 h (i.e. total dose of 0.4 mg) completely prevented this recovery. Prolactin concentration was also reduced after the s.c. implantation of a solid pellet of bromocriptine. Once daily injections of bromocriptine for 7 days starting 1-day prepartum (early lactation group) or on day 5 of lactation (established lactation group) significantly reduced milk yield, assessed from the weight gain of the litter or estimated by a tritiated water dilution procedure. In early lactation the degree of inhibition was positively related to the size of the suckling litter (three pups, no inhibition; nine pups, 16.4% inhibition; 14 pups, 40.8% inhibition), but in established lactation an inhibition of 25-30% was observed, regardless of litter size. Twice-daily injections and s.c. implants of bromocriptine both reduced milk yield by a greater amount (approximately 45%), but in no case was secretion completely suppressed. The inhibitory effect of bromocriptine was prevented by the simultaneous administration of ovine prolactin (0.4 mg). Lactogenesis was apparently not affected by bromocriptine treatment. At the end of the early lactation treatment period, the mammary glands of bromocriptine-treated animals were significantly smaller and contained fewer cells than those of controls, but this was not the case in animals treated during established lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Galactopoietic and mammogenic effects of long-term treatment with bovine growth hormone and thrice daily milking in goats.

Starting in mid-lactation, goats were treated daily for 22 weeks with 0.15 mg recombinant bovine GH (bGH)/kg, or an equivalent volume of vehicle. One gland of each goat was milked thrice daily throughout treatment, the other twice daily. Mammary differentiation was studied in biopsy samples obtained before treatment and after 3 and 22 weeks of treatment, by determination of in-vitro synthesis rates of milk constituents and measurement of enzyme activities. Mammary growth was measured using a whole-body imaging technique (magnetic resonance imaging; MRI). bGH caused an immediate and sustained increase in milk yield of approximately 23% overall, whilst the glands milked thrice daily produced approximately 14% more than the control glands milked twice daily. The effects of the combined treatment were additive, but not synergistic. A synergistic effect of the combined treatment resulted in a significant improvement in lactation persistency. A stimulatory effect of milking frequency on mammary enzyme activities was evident only in bGH-treated goats at 3 weeks, but in both groups at 22 weeks. Synthesis rates of casein and lactose were increased at 3 weeks only by the combined treatment. Thus bGH accelerated or augmented the differentiative response to thrice daily milking. Mammary parenchyma volume, estimated by MRI, increased significantly during the first 12 weeks of bGH treatment and remained higher throughout the rest of the treatment period. Cell number was estimated from parenchyma volume and DNA concentration; this decreased significantly in the controls between weeks 1 and 22, but remained constant in the bGH group. In nine of the ten goats, parenchyma volume and cell number increased in the gland milked thrice daily relative to the control gland milked twice daily during the course of the experiment. Thus bGH stimulated growth of the mammary gland over and above that induced by the frequent milking. The absence of any detectable increase in thymidine incorporation suggests that this growth consisted of cellular hypertrophy rather than hyperplasia.

Autocrine regulation of casein turnover in goat mammary explants.

Local feedback control of milk protein secretion was investigated in goat mammary explants by measuring degradation of newly synthesized casein in the presence of a goat milk whey fraction. Reduced net synthesis of [3H]casein in the presence of the milk fraction was due, at least in part, to its degradation during secretion, suggesting that this process is under autocrine regulation.

Effects of an antiserum to rat growth hormone on lactation in the rat.

A highly specific antiserum to rat GH (anti-rGH) was used to assess the role of GH in lactation in the rat. When administered alone, anti-rGH had no effect on litter weight gain, whereas bromocriptine reduced serum prolactin concentrations and litter weight gain for up to 7 days when given on day 4 of lactation. When bromocriptine and anti-rGH were given in combination, however, litter weight gain declined even more dramatically so that pups were receiving virtually no milk 2-3 days after treatment. Daily litter exchange failed to prevent this effect. Concurrent injections of highly purified GH (prolactin contamination undetectable) prevented the dramatic decline in litter weight gain induced by combined bromocriptine and anti-rGH treatment, so that these litters grew as well as those receiving bromocriptine alone. Growth hormone did not act by influencing serum prolactin concentrations, which remained low during GH therapy. Direct effects of anti-rGH or GH on the pups (transferred through the milk) were ruled out since virtually identical results were obtained when milk yield was estimated during a 30-min suckling period after a 3-h separation of mother and pups. Lactation had virtually ceased 3 days after treatment with both bromocriptine and anti-rGH, but it could be reinitiated by a single injection of prolactin or GH, and subsequent recovery was virtually complete. The results of this study show that prolactin can maintain a full milk yield in the absence of GH, milk yield is reduced by approximately 50% in the absence of prolactin and milk yield is totally stopped in the absence of prolactin and GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine profiles of cows undergoing extended lactation in relation to the control of lactation persistency.

We conducted an experiment in dairy cows investigating the effects of calving season, milking frequency and nutrition on lactation persistency. Cows calved in the Spring (n=12) or Winter (n=12). Commencing in lactation week 9 one udder-half of each cow was milked thrice-daily and half of each calving group received additional concentrate at a fixed rate of 3kg per day above that of the control cows. As reported elsewhere, between lactation weeks 9 and 33 persistency (measured as the slope of decline in milk yield) was significantly improved by frequent milking (P<0.001), by calving in the Winter (P<0.001) and by additional concentrate (P<0.05). The cows were rebred after week 33. When analysis of persistency was extended up to week 20 of the recurring pregnancy only the frequency effect remained significant. Persistency was unaffected by the pregnancy up until pregnancy week 20 but was then greatly reduced (P<0.001). In this paper we report hormone concentrations. GH was unaffected by nutrition but was consistently elevated in the Winter calving group relative to the Spring. IGF1 and prolactin were both unaffected by nutrition and calving season, IGF1 tended to increase as lactation progressed but changes in prolactin were related to time of year more than stage of lactation. Insulin was not affected by nutrition and was lower in Winter calvers, but only during early lactation. Prior to rebreeding, lactation persistency was correlated (slightly) with [GH] but not with [IGF1] or [insulin] and was correlated significantly with changes in GH, IGF1 (both positive) and insulin (negative). In conclusion, whilst bovine lactation persistency is plastic and amenable to beneficial manipulation, the details of its endocrine control remain to be elucidated.

Growth hormone and mammary development.

Classic studies in rodents conducted in the 1950s showed that growth hormone (GH) is essential for mammary development both in the pubertal phase and during pregnancy. Since then, a considerable number of experiments have been carried out in ruminants to investigate the role of GH for regulation of normal mammary development and to examine the possibility of enhancing mammary growth by administration of GH. The available evidence demonstrates that GH treatment stimulates mammary growth before puberty, but the data do not convincingly support the idea that the effect is translated into increased milk yield. GH treatment during late pregnancy seems to stimulate both mammary growth and milk yield during lactation. The limited data concerning the effect of GH on mammary growth during lactation indicate that mammary growth is unaffected by GH treatment in early lactation, whereas GH seems to increase the amount of mammary parenchyma in mid-lactation. The mechanism of action of GH remains a puzzle, but the effect of exogenous GH most likely involves insulin-like growth factor-I (IGF-I). Full understanding of the role of endogenous GH for regulation of normal mammary development requires more knowledge about the interaction between GH and IGF-I and the interplay between the GH-IGF-I axis and locally produced factors, including receptors, binding proteins, and growth factors.

A comparison of hoof lesions and behaviour in pregnant and early lactation heifers at housing.

Lameness, hoof lesion development and behaviour were compared for two groups of 10 heifers: one in early pregnancy (PH), the other in early lactation (LH). Both groups were housed in the summer in cubicles under identical conditions. Behavioural observations commenced immediately after housing, and then at 2, 4 and 6 weeks post-housing. Locomotion scores were assessed weekly, and feet were examined for lesions at approximately -1, 0, 1 and 2 months after housing. All four feet were photographed, lesions were scored subjectively for severity, and lesion size and position were estimated using image analysis techniques. LH already had greater total lesion scores before housing. More severe linear lesions in the LH group were associated with reduced lying, and less idling, increased standing in cubicles and more disturbed lying behaviour.

Effects of long-term thrice-daily milking on mammary enzyme activity, cell population and milk yield in the goat.

In two separate experiments, lactating goats were milked unilaterally thrice daily instead of twice daily for periods of 13 and 37 wk, starting at 2 to 5 wk after parturition. The other gland was milked twice daily throughout. In both experiments thrice-daily milking increased milk yield significantly. In the first experiment, after 37 wk the amounts of RNA and DNA, rate of cell proliferation and activities of several enzymes per cell were greater in the thrice-milked gland (P less than .05). In the second experiment, after 9 or 10 d of thrice-daily milking the activities of several key mammary enzymes per cell measured in biopsy samples had increased in the thrice-milked but not the twice-milked gland. After 13 wk of thrice-daily milking, the DNA content of the glands and the activity per cell of the key enzymes was the same in both glands, showing that the synthetic capacity of the gland was being used more effectively in the treated gland. These results indicate that several mechanisms contributed to the increased milk yield induced by thrice-daily milking.