Effects of intramammary infusions of casein hydrolysate,ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70 mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7 g), lactose (5.1 g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na⁺ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K⁺ concentrations decreased. As involution advanced, the Na⁺:K⁺ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na⁺ concentration and in the Na⁺:K⁺ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.     

Effect of intramammary infusion of chitosan hydrogels at drying-off on bovine mammary gland involution

The transition from lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when the involution of the mammary gland is completed. Accordingly, approaches that speed up the involution process after drying-off could reduce the incidence of mastitis. The current study aimed to develop a biological response modifier that could be injected into cow teats to promote immune cell migration and speed up involution. Chitosan, a natural polysaccharide derived from chitin, is able to trigger host innate immunity. We developed 2 formulations made from either high- or low-viscosity chitosan. Both are liquid at room temperature but form a hydrogel at body temperature. In the first experiment, each udder quarter of 7 Holstein cows in late lactation was randomly assigned at drying-off to receive one of the following intramammary infusions: 2.5 or 5 mL of 5% (wt/vol) low-viscosity chitosan hydrogel, 5 mL of 5% high-viscosity chitosan hydrogel, or 5 mL of water. Milk (mammary secretion) samples were collected from each quarter on d ?4, ?1 (drying-off), 1, 3, 5, 7, and 10. Milk somatic cell counts and the concentrations of involution markers such as BSA, lactate dehydrogenase, and lactoferrin were measured in each sample. In comparison with the control, the chitosan hydrogel infusions significantly hastened the increases in somatic cell counts, BSA and lactoferrin concentrations, and lactate dehydrogenase activity in mammary secretions. No major differences between sources or volumes of chitosan were observed for the measured parameters. The compatibility of this approach with an internal teat sealant was verified in the second experiment. Each udder quarter of 8 Holstein cows was randomly assigned at drying-off to receive one of the following intramammary infusions: 5 mL of 2% low-viscosity chitosan hydrogel, 4 g of an internal teat sealant, a combination of sealant and chitosan, or 5 mL of water. Milk (mammary secretion) samples were collected from each quarter on d ?4, ?1 (drying-off), 5, and 10 to measure involution markers. These results suggest that chitosan hydrogel infusion hastened mammary gland involution and activate immune response, which may reduce the risk of acquiring new intramammary infections during the drying-off period. Those results were not affected by the presence of the teat sealant, showing that both approaches are fully compatible and could be used in combination.     

Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows

Milk stasis triggers local stimuli, which make the tight junctions leak and trigger involution. The aim of the study was to test the hypothesis that casein hydrolyzates compromise tight junction integrity and dry-off milk secretion in dairy cows. Six repeated doses of casein hydrolyzates after each milking during 3 d caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion. No such changes were recorded in the control glands that had been treated with nonhydrolyzed casein. Treatment with casein hydrolyzates disturbed tight junction integrity within 8 h (as indicated by changes in Na+ and K+ concentrations), reduced the concentrations of lactose precipitously, activated the plasmin activator-plasminogen-plasmin system, and induced the secretion of immunoglobulin type G and lactoferrin. At the end of the 3-d treatments, we stopped milking the experimental and control glands. Milk composition 19 d later was similar in the experimental and control glands and was consistent with the composition expected in fully involuted glands. We conclude that casein hydrolyzates are among the milk-borne factors that cause the disruption of tight junction integrity and induce involution in cows. The process induced by casein hydrolyzate was more rapid and synchronized than the involution induced at drying-off.     

Early mammary gland metabolic and immune responses during natural-like and forceful drying-off in high-yielding dairy cows

The present work compared metabolic and immune responses in genetically high-producing cows that produced a low amount of milk before expected involution and in cows with the same genetic potential that produced copious amounts of milk before their scheduled drying-off. Ten multiparous lactating Israeli Holstein cows producing approximately 10,500 L in the current lactation, without bacterial infection and scheduled for drying-off approximately 60 d before their expected parturition, were studied. Five of the cows that exhibited a sharp, spontaneous reduction in milk yield at the end of their lactation and produced less than ~14 L/d were defined as cows approaching natural involution (ANI), and 5 cows that produced between 25 and 35 L/d were defined as cows approaching forced involution (AFI). Three days before scheduled drying-off, milking was stopped and milk samples were collected from each quarter. After milking cessation, only modest swelling was observed in the udders of the ANI cows. In the ANI cows, lactose and fat concentrations decreased and the fat:lactose concentration ratio indicated that on d 1 and 2 fat concentrations decreased faster than lactose concentration, whereas on d 3, the rate of reduction was about the same for lactose and fat. In contrast, in AFI cows, fat concentrations increased on d 1 and the fat:lactose ratio indicated that changes in fat secretion were minor compared with those of lactose secretion. Rennet clotting time of milk after drying-off in the ANI cows increased, whereas curd firmness decreased rapidly, such that mammary secretions did not coagulate on d 3. In the AFI cows, such significant changes were observed only on d 3. The inflammatory response increased in both groups, but at each stage the increase was greater in ANI cows than in AFI cows. On d 1, the increase in leukocyte numbers in the ANI cows was made up of mononuclear cells (i.e., T lymphocytes and macrophages). In contrast, in the AFI cows, we observed a marked increase in leukocyte numbers, mainly in the form of polymorphonuclear cells. Our data indicate that the abrupt mammary involution induced in AFI cows provoked signs of distress, which were associated with neutrophilia in milk. In contrast, in the ANI cows, cessation of milking occurred without evidence of engorgement of the udder. Physiological differences in ANI and AFI cows are distinct and are reflected in the differences in the leukocyte populations in milk.     

Effects of photoperiod modulation and melatonin feeding around drying-off on bovine mammary gland involution

The risk for a dairy cow to acquire new intramammary infections is high during the transition from lactation to the dry period, because of udder engorgement and altered immune functions. Once the gland is fully involuted, it becomes much more resistant to intramammary infections. Therefore, strategies to depress milk yield before drying-off and accelerate the involution process after drying-off could be beneficial for udder health. The objective of this study was to assess the effect of photoperiod manipulation and melatonin feeding from 14 d before to 14 d after drying-off on the speed of the involution process. Thirty Holstein cows in late lactation were randomly allocated to one of the following treatments: (1) a long-day photoperiod (16 h of light: 8 h of darkness), (2) a short-day photoperiod (8 h of light: 16 h of darkness), and (3) a long-day photoperiod supplemented by melatonin feeding (4 mg/kg of body weight). Milk and blood samples were collected on d ?26, ?19, ?12, ?5, ?1, 1, 3, 5, 7, 10, and 14 relative to the last milking to determine concentrations of mammary gland involution markers and serum prolactin. Additional blood samples were taken around milking on d ?15, before the start of the treatments, and on d ?1, before drying-off, to evaluate the treatment effects on milking-induced prolactin release. The short-day photoperiod slightly decreased milk production and basal prolactin secretion during the dry period. The milking-induced prolactin surge was smaller on d ?1 than on d ?15 regardless of the treatments. Lactoferrin concentration, somatic cell count, and BSA concentration as well as matrix metalloproteinase-2 and -9 activities increased in mammary secretions during the first 2 wk of the dry period, whereas milk citrate concentration and the citrate:lactoferrin molar ratio decreased. The rates of change of these parameters were not significantly affected by the treatments. The long-day photoperiod supplemented by melatonin feeding did not affect milk production, prolactin secretion, or mammary gland involution. Under the conditions in this study, photoperiod modulation and melatonin feeding did not appear to affect the rate of mammary gland involution.     

Influence of the lactoperoxidase system on susceptibility of the udder to Streptococcus uberis infection

Lactoperoxidase (LP), thiocyanate (SCN-), pH and somatic cell counts (SCC) were measured in mammary secretions from 20 cows collected 14 d before drying-off, 7 and 21 d after drying-off, and 3-18 d postcalving. The inhibitory activity of the secretions on Streptococcus uberis was determined and the susceptibility of the udder to infection by this organism was tested by intramammary infusion of 250 colony forming units at the above stages. LP, SCN-, pH and SCC increased during involution and fell postcalving. The secretions collected before drying-off, 7 d after drying-off and postcalving inhibited growth of Str. uberis.; those collected 21 d after drying-off did not. Inhibitory activity in pre-drying-off secretions was destroyed by heating and restored by addition of LP, glucose and glucose oxidase, but addition of these substances to secretion 21 d after drying-off did not provide a full inhibitory system. The growth of Str. uberis in the secretions was correlated with intramammary susceptibility, since challenges with Str. uberis at 14 d before drying-off, at 7 and 21 d after drying-off and postcalving led to 43.8, 25.0, 81.3 and 37.5% of quarters becoming infected. It is suggested that the LP/SCN-/H2O2 system plays a role in protecting the lactating mammary gland from infection with Str. uberis but becomes ineffective as involution progresses.     

Effects of feed restriction and prolactin-release inhibition at drying off on metabolism and mammary gland involution in cows

A cow’s risk of acquiring a new intramammary infection during the dry period increases with milk production at drying off and decreases as mammary gland involution progresses. A method commonly used to reduce milk production is a drastic reduction in feed supply in the days that precede drying off. Milk production can also be reduced by inhibiting the lactogenic signal driven by prolactin (PRL). This study aimed to compare the effects of these 2drying-off procedures on metabolism, immunity, and mammary gland involution in cows. A total of 24Holstein cows in late lactation were assigned to 1 of 3treatments based on milk yield, somatic cell count, and parity. The cows were fed a lactation diet until drying off (control; n = 8), only dry hay during the last 5 d before drying off (DH; n = 8), or the same lactation diet as the control cows but with twice-daily i.m. injections of 4 mg of quinagolide, a specific inhibitor of PRL release, from 5 d before drying off until 13 d after (QN; n = 8). Quinagolide induced a decrease in PRL concentration in blood and in milk and mammary secretions on all the injection days. Interestingly, PRL was also depressed in the blood and milk of the hay-fed cows before drying off. Both the QN and DH treatments induced a decrease in milk production, which averaged 17.9 and 10.1 kg/d for the QN and DH cows, respectively, at drying off in comparison with 24.8 kg/d for the control cows. Both BSA concentration and Na⁺-to-K⁺ ratio increased faster in the mammary secretions of both the DH and QN cows than in those of the control cows, whereas citrate-to-lactoferrin ratio, another indicator of involution rate, decreased faster. The DH treatment decreased blood concentrations of glucose and most amino acids and increased blood concentrations of β-hydroxybutyrate and nonesterified fatty acids. Quinagolide increased blood glucose but did not affect the other metabolites. The serum harvested on d−1 from the hay-fed cows reduced peripheral blood mononuclear cell proliferation and IL-4 production, whereas the serum from the quinagolide-treated cows had no effect. In conclusion, this experiment shows that PRL-release inhibition could be a new alternative for reducing milk production before drying off and for hastening mammary gland involution without disturbing the metabolism of the cow.     

Lactoferrin concentration during involution of the bovine mammary gland.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.     

Effects of secretion removal on bovine mammary gland function following an extended milk stasis.

The objective of this study was to determine whether lactation function could be reinitiated after a period of extended milk stasis. Involution was induced by milk stasis in lactating Holstein cows for a period of 11 d. On d 11, one side of the mammary gland was milked twice daily for 3 d. The contralateral side remained unmilked for the 14-d experimental period. Cows were slaughtered, and mammary tissue was collected from both udder halves for further analysis. Mammary secretion volume was partially restored in the milked udder half, but reestablished milk yields were variable among cows. A partial recovery of lactation function was further indicated by elevated levels of lactose and protein profiles resembling milk in mammary secretions from the milked glands. Lactose and protein profiles from the unmilked glands were similar to those of glands undergoing involution. Lactoferrin levels were elevated in secretions from the milked and unmilked udder halves. Casein and lactoferrin synthesis by mammary explants and beta-casein and lactoferrin mRNA abundance in mammary tissues corresponded to protein profiles from milked and unmilked mammary secretions. alpha-Lactalbumin mRNA was variable but was more abundant in the milked glands compared with the unmilked glands. Lectin fluorescence microscopy for soybean agglutinin preferentially stained the apical surface of the mammary epithelial cells from the milked glands. Staining was absent in the unmilked glands and suggested resumption of lactation function in all such milked glands. These results suggest that mammary involution can be partially reversible after 11 d of milk stasis.     

Short communication: suppression of estrous cycles in lactating cows has no effect on milk production

The decline in milk yield observed after peak production in dairy animals results from apoptotic death of mammary epithelial cells. In cows, this decrease in milk yield can be accelerated by injection of 17β-estradiol, thus evoking a possible role of estrogens in the regulation of bovine mammary gland involution. In nonpregnant cows, mammary involution could be induced or enhanced by the return of estrous cycles and the accompanying cyclic peaks of estrogen concentration in the serum of lactating animals. To test this hypothesis, we inserted implants of a GnRH agonist, deslorelin, in an ear of each cow (n = 10) on d 10 and 100 of lactation, to temporarily suppress the return of ovarian cycles. Cows were studied from calving to d 210 of lactation. Deslorelin had no impact on feed intake or animal health. Deslorelin significantly reduced serum concentrations of 17β-estradiol and progesterone as compared with untreated cows (n = 10). Deslorelin had no effect on milk fat and protein, whereas milk lactose content was lower in treated cows than in control cows on d 100 of lactation. Finally, there was no difference in milk production between the 2 groups of cows. These results are consistent with previous observations that showed that delaying estrous cycles after calving had no effect on milk yield and they extend those observations to late lactation. Based on milk production data, the estrogen profiles associated with recurring estrous cycles apparently do not cause bovine mammary tissue to undergo gradual involution.     

Role of tissue remodeling in mammary epithelial cell proliferation and morphogenesis.

Lactation is a physiological process characterized by the secretion of large quantities of protein, carbohydrate, and lipid. To achieve the production, the mammary gland must grow and then differentiate; both processes require extensive tissue remodeling. Remodeling begins with a carefully controlled proteolysis of the extracellular matrix and cell-cell adhesion proteins. Plasmin is a serine protease that has been implicated in the tissue remodeling associated with the declining phase of lactation and mammary involution. As lactation progresses, the quantity of plasmin activity increases within the mammary tissue and milk. This has led to the hypothesis that gradual involution results from progressive tissue remodeling. Hormonal attenuation of gradual involution by bST would slow tissue remodeling and would be permissive for lactation. In vitro results indicate that insulin-like growth factor-I impairs the secretion of plasminogen activator by bovine mammary epithelial cells. As such, a mechanism of action for bST exists.     

Invited review: Accelerating mammary gland involution after drying-off in dairy cattle

Bovine mammary gland involution, as a part of the reproductive cycle in dairy cows, is a very important remodeling transformation of the mammary gland for the subsequent lactation. There is considerable incentive to accelerate mammary gland involution to improve udder health, shorten the dry period, and simplify the management process by reducing dietary changes. The complex process of mammary involution is characterized by morphological changes in the epithelial cells and mammary tissue, changes in the composition of mammary secretions, and changes in the integrity of tight junctions. Involution is facilitated by elements of the immune system and several types of proteases and is coordinated by various types of hormones. This review first describes the involution process and then argues for the need to accelerate it. Last, this review focuses on various intervention methods for accelerating involution. Our aim is to provide a comprehensive overview of bovine mammary gland involution as well as potential techniques and new opinions for dry cow management.     

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.     

beta-Lactoglobulin and alpha-lactalbumin in mammary secretions during the dry period: parallelism of concentration changes

Proteins in mammary secretions were examined during the dry period of four cows. Polyacrylamide gel electrophoresis was used to determine relative changes in the electrophoretic profiles of whey proteins in the mammary secretions. Concentrations of beta-lactoglobulin and alpha-lactalbumin in dry cow secretions were determined by immunoassay. Concentrations of both proteins changed in parallel during the dry period. Concentrations of beta-lactoglobulin and alpha-lactalbumin decreased during the first 30 d of the dry period but generally remained above .1 mg/ml secretion. A transient increase in beta-lactoglobulin and alpha-lactalbumin concentrations occurred at about 40 d after drying-off, prior to the prepartum increase.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

Enzyme-linked immunosorbent assays of bovine lactoferrin and a 39-kilodalton protein found in mammary secretions during involution

Enzyme-linked immunosorbent assays were developed for two proteins found in mammary secretions during the non-lactating period: lactoferrin and a 39-kdal protein. Minimum detectable concentrations were .12 ng/ml for lactoferrin and .0037 ng/ml for the 39-kdal protein. Standard curves were linear in the ranges of .4 to 100 ng/ml for lactoferrin and 2.4 to 30 ng/ml for the 39-kdal protein. The immunoassays were used to measure concentrations of the proteins in mammary secretions during involution. From the last day of milking to d 30 of involution, mean concentrations of lactoferrin and the 39-kdal protein increased from .82 +/- .35 to 78.5 +/- 12.43 mg/ml and from 11.8 +/- 3.8 to 84.4 +/- 21.1 micrograms/ml, respectively. The immunoassays described will be valuable for further studies on the synthesis and secretion of these proteins during mammary involution.     

Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows

Bovine somatotropin (bST) increases milk production in lactating cows through its effect on nutrient partition and maintenance of mammary cell function. A positive relationship between bST treatment and abundance of β-casein mRNA in mammary tissues from lactating cows was hypothesized. In mammary tissue isolated from 14 midlactation Holstein cows, β-casein mRNA was 35.4% higher among 7 cows receiving continuous bST infusions at 29 mg/d for 63 d compared with tissue from 7 untreated control cows. To investigate whether increased β-casein mRNA resulted from a direct effect of bST on the mammary gland, explants of mammary tissue from other lactating cows that had not received bST were incubated with bST and prolactin in 2 experiments. Mammary explant cultures taken from 2 lactating cows that had not been milked for 48 h were supplemented with either prolactin or bST. Both prolactin and bST stimulated higher levels of β-casein mRNA in the mammary explants compared with their non-supplemented counterparts. Explant cultures from 4 additional lactating cows were prepared from rear quarter mammary tissue subjected to milking intervals of 6 h for right rear quarters or 20 h for left rear quarters. Both bST- and prolactin-mediated increases in β-casein mRNA were dependent on milking intervals. That is, levels of β-casein mRNA were increased by bST or prolactin supplementation in explants isolated from the mammary quarters biopsied 20 h after milking but not for those biopsied at 6 h after milking. Results are consistent with a potential role for bST in up-regulating or sparing β-casein mRNA levels in lactating bovine mammary tissue in a manner similar to prolactin.