Quantification of mammary gland tissue size and composition changes after weaning in sows

The objectives of this study were to characterize the tissue compositional changes in porcine mammary glands after weaning and to determine whether administration of estradiol alters the profile of these tissue changes. Forty-five primiparous sows were assigned randomly to one of two treatment groups after weaning, control or estrogen treated. Estrogen-treated sows received twice-daily injections of estradiol-17beta (0.125 mg/kg of BW); control sows received vehicle injections. Sows were weaned at d 21 of lactation and killed on either d 0 (d of weaning; n = 5) or on d 2, 3, 4, 5, or 7 after weaning (n = 4 per treatment on each day). Teat order relative to suckling behavior was observed on the day before weaning to determine which mammary glands the piglets suckled. Suckled and non-suckled glands were identified from the teat order observation, and individual mammary glands were collected at slaughter. Mammary glands were trimmed of skin and extraneous fat pad, individually weighed, and bisected to measure cross-sectional area. The remaining half of each gland was ground and stored at -20 degrees C for chemical analyses. Frozen tissue was used for measuring tissue DNA, DM, protein, fat, and ash contents. Suckled mammary glands of sows undergo significant and dramatic changes during the initial 7 d after weaning, with significant changes occurring even by d 2 after weaning. Mean cross-sectional area of parenchymal tissue in suckled mammary glands decreased from 59.7 +/- 2.1 cm2 on the day of weaning to 26.8 +/- 2.3 cm2 by d 7 after weaning (P < 0.0001). Mammary gland wet weight decreased from 485.9 +/- 22.0 g on the day of weaning to 151.5 +/- 24.8 g by d 7 after weaning (P < 0.0001), whereas DNA decreased from 838.8 +/- 46.2 g on the day of weaning to 278.4 +/- 52.5 g by d 7 after weaning (P < 0.0001). The changes in gland wet weight and DNA during the period of mammary gland involution in the sow represent loses of over two-thirds of the parenchymal mass and nearly two-thirds of the cells that were present on the day of weaning. Estrogen treatment did not affect overall mammary involution during the first 7 d after weaning. Mammary glands that were not suckled during lactation had no further loss of parenchymal tissue during the first 7 d after weaning. Mammary gland involution in the sow is a rapid process and is probably irreversible within 2 or 3 d after weaning.     

Effects of steroid hormone treatment on mammary development in prepubertal heifers

The objective of this study was to determine the effects of steroid hormone implantation in heifer calves on the ability of mammary tissue to develop subsequently in organ culture. Twenty-four calves were paired by date of birth and assigned to groups (eight calves/group). At 4, 7, or 10 mo of age, calves were implanted subcutaneously (s.c.) with pellets containing cholesterol or cholesterol, 17β-estradiol, and progesterone for 9 or 18 d. The calves were euthanized and uteri and mammary glands were removed and weighed. Slices of mammary parenchymal tissue were incubated for 5 d at 37°C in a 50% O₂, 5% CO₂ humidified atmosphere in Waymouth’s 752/liter medium supplemented with insulin (5.0 μg/ml) or lactogenic hormone complex insulin (5.0 μg/ml), aldosterone (0.1 μg/ml), hydrocortisone (0.1 μg/ml), and prolactin (1.0 μg/ml) in the presence or absence of epidermal growth factor (EGF) (0.06 μg/ml) to promote lobulo-alveolar development. Tissue sections were stained and mounted on slides for morphologic and histologic analysis or prepared to evaluate expression of β-casein mRNA. There were no morphologic differences in slices from calf mammary tissues despite age, steroid hormone priming, or hormones used in tissue culture. The 4-mo-old calves required steroid priming followed by incubation of the tissue slices with the lactogenic complex with or without epidermal growth factor to induce cytological changes associated with lactogenesis but did not express β-casein mRNA. At 7 mo of age, steroid hormone priming was not necessary for induction of alveolar formation and secretion. Incubation of the tissue slices from 7-mo-old calves with the lactogenic complex was sufficient to induce alveolar formation and secretion. However, β-casein mRNA was not expressed. At 10 mo of age, exposure of tissue from calves to the lactogenic hormones caused histologic changes reminiscent of the ability to secrete milk regardless of hormone priming. However, estrogen and progesterone priming was necessary before incubation of the tissue slices with the lactogenic hormones to induce β-casein mRNA expression. When epidermal growth factor was added to the lactogenic hormone complex, β-casein mRNA expression decreased. These data support the concept that there is a sequential development of responsiveness of the mammary gland to various hormones. By 10 mo of age, prepubertal heifers reach a stage of maturity where steroid hormone priming followed by incubation of tissue slices with the lactogenic hormones is sufficient to induce both structural and functional differentiation.     

Ultrasonographic assessment of the feline mammary gland

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5-9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6-9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.     

Relationship between weaning and secretion of luteinizing hormone, cortisol and transcortin in beef cows

The effects of suckling on secretion of luteinizing hormone, cortisol and transcortin were investigated in anovulatory postpartum cows. On d 35 postpartum, calves were separated from 12 cows to prevent suckling and eight calves continued to suckle their dams ad libitum. Between 35 and 41 d postpartum, samples of jugular blood were collected every 15 min for two periods of 6 h/d. In non-suckled cows, frequency of pulses and basal luteinizing hormone increased but amplitude of pulses did not change. Concentrations of total cortisol in serum of cows were not altered during 3 d after weaning calves and did not differ among intervals before, during and after a suckling event. Affinity of transcortin for cortisol was not affected by postpartum interval or treatment. Capacity of transcortin to bind cortisol tended to increase after weaning. We found no evidence to support the hypothesis that suckling reduces binding capacity of transcortin or increases unbound cortisol. Differences in preovulatory secretion of luteinizing hormone between suckled and non-suckled cows could not be accounted for by differences in secretion of cortisol. In beef cows that are fed to satisfy requirements for energy and have average body condition, we conclude that negative modulation of luteinizing hormone by suckling is not mediated by cortisol.     

Effect of suckling on the reproductive performance and metabolic status of obese Japanese black cattle during the early postpartum period

The aim of our study was to investigate the effects of suckling on reproductive performance and metabolic status of obese (mean body condition score of more than 4.0 on a scale of 1-5) maternal Japanese Black cows during early postpartum period. We used 7 postpartum Japanese Black cattle. Four cows were suckled ad libitum (suckled) until completion of their first artificial insemination (AI), while 3 cows were not suckled at all because they were separated from their calves immediately after parturition (non-suckled). Body weight and plasma concentrations of metabolites and hormones were measured from wk 1 to 9 postpartum. Ovarian activity was detected using plasma progesterone concentration, and all cows received their first AI after application of the Ovsynch protocol at approximately 4 months postpartum. Although body weights of non-suckled cows increased during experimental period (P<0.05), those of suckled cows remained unchanged. Plasma concentrations of glucose of non-suckled cows were higher at wk 2 postpartum (P<0.05) and their levels of non-esterified fatty acid tended to be lower at wk 1 and 2 postpartum compared with suckled cows (P<0.1); however, these differences between groups were not observed with progression of postpartum period. In addition, plasma insulin concentrations of non-suckled cows were higher than those of suckled cows during experimental period (P<0.05). During sampling period (wk 0 to 9 postpartum), onset of normal ovarian cycle was observed in all non-suckled and 2 of 4 suckled cows, and it was delayed in other 2 suckled cows compared with non-suckled cows; however, 3 suckled cows conceived at the first AI after application of the Ovsynch protocol; none of non-suckled cows conceived at this time. Overall, we suggest that suckling seems to reduce increase of body weight after parturition, although it does not improve obesity, and influences conception despite delay in resumption of normal ovarian cyclicity in obese Japanese Black cows.     

Changes in tissue composition associated with mammary gland growth during lactation in sows.

Twenty-four primiparous sows were used to determine the extent of mammary gland growth during lactation. Litter size was set to nine or 10 pigs immediately after birth. Sows were slaughtered in groups representing d 0 (within 12 h after farrowing), 5, 10, 14, 21, and 28 of lactation. Sows were provided 17.5 Mcal ME and 65 g of lysine per day during lactation. Mammary glands were collected at slaughter and trimmed of skin and extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, and ground for chemical analysis. Dry matter content, dry fat-free tissue (DFFT) content, protein content, amino acids composition, ash content, and DNA content were measured. Only glands known to have been suckled were included in these data. Wet and dry tissue weight; cross-sectional area; and the amount of DFFT, tissue protein, and amino acids in each suckled mammary gland increased (P < .05) during lactation to a peak on d 21. Fat percentage of each suckled gland declined (P < .05) and the percentage of protein and DFFT increased (P < .05) as lactation progressed. These results suggest that hypertrophy occurred in the tissue during lactation. There was a linear increase in the amount and percentage of DNA during lactation (P < .05), suggesting hyperplasia of the mammary tissue. Mammary tissue growth continues in suckled glands during lactation in sows, with gland wet weight increased by 55% and total gland DNA increased by 100% between d 5 and 21 of lactation.     

Effect of nutrient intake on mammary gland growth in lactating sows

Sixty-one primiparous sows were used to determine the response of mammary gland growth to different energy and protein intakes during lactation. After birth, litter size was set to 9 or 10 pigs. Sows were slaughtered at selected times up to 30 d of lactation. Individual sows were fed one of four diets that were combinations of different amounts of energy and protein (3.0 Mcal ME and 8.0 g lysine/kg diet; 3.0 Mcal ME and 16.2 g lysine/kg diet; 3.5 Mcal ME and 6.4 g lysine/kg diet; or 3.5 Mcal ME and 13.0 g lysine/kg diet). Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, ground, and stored at -20 degrees C for chemical analysis. Frozen, ground tissue was used to determine dry matter, dry fat-free tissue (DFFT), total tissue protein, ash, and DNA content. Only glands known to have been suckled were included in this data set. Response surface regression was used for statistical analysis. The percentage of protein, fat, ash, and DNA in each suckled mammary gland was affected only by total energy intake (P<.05). The percentage of dry tissue and fat decreased as the total energy consumed during lactation increased, whereas the percentage of protein and DFFT increased as total energy intake increased. There were quadratic effects (P<.05) of both total energy and protein intake on wet weight, dry weight, protein amount, DFFT amount, and DNA amount of each suckled mammary gland during lactation. This study shows that mammary gland growth is affected by nutrient intake during lactation. The weight of suckled mammary glands and the amount of mammary tissue protein, DFFT, and total DNA were maximal on d 27.5 of lactation when sows had consumed an average of 16.9 Mcal of ME and 55 g of lysine per day during lactation. Provision of adequate amounts of nutrients to sows during lactation is important for achieving maximal growth of mammary glands and maximal milk production.     

Evidence of a Direct Role for Growth Hormone (GH) in Mammary Gland Proliferation and Lactation

A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in ductal and alveolar epithelial cells from rat and rabbit mammary glands by immunohistochemistry. Intense immunoreactivity was present in membrane, cytoplasm and some nuclei of epithelial cells during proliferation and lactation. Receptor expression decreased during weaning and was absent or weak in regressive mammary glands. Immunoreactivity was weak in ductal epithelial cells from virgin adult animals. Pronounced expression of GH receptor/binding protein was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the existence of a soluble form on the GH receptor, namely the growth hormone binding protein derived from the membrane receptor by cleavage. Primary localization of the receptor in proliferating and lactating epithelial cells suggests that the rat and rabbit mammary gland is a GH target tissue. This finding is in contradiction to both classical GH action and the somatomedin hypothesis and challenges the widely held view that GH has no direct influence on mammary growth and function.     

Quantitation of calmodulin in the mammary gland of the lactating sow

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

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 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.     

Expression of estrogen receptor 1 and progesterone receptor in primary goat mammary epithelial cells

The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR) was quantified in mammary tissue‐derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up‐regulation of ESR1 (1.36‐ to 12.35‐fold) and in down‐regulation of PGR (‐2.53‐ to ‐3.62‐fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER‐α) positive cells do not exclusively belong to the luminal lineage and that ER‐α and PGR can be expressed individually or co‐expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone‐responsive and represent a useful surrogate for mammary tissue in research experiments.     

Morphometric analysis of involuting bovine mammary tissue after 21 or 42 days on non-suckling

Mammary gland involution was morphologically evaluated 21 or 42 d after prevention of suckling of one udder half in 10 crossbred beef cows. Parenchymal tissue was taken from lower, middle and upper zones of each quarter from the teat to the ventral body wall. Udder halves, trimmed of extraparenchymal tissue, were weighed and used for DNA determination. DNA content was reduced 50 and 64% after 21 and 42 d of involution. However, the percentage of tissue occupied by epithelium was similar in suckled and nonsuckled glands. Well-differentiated cells, typical of suckled glands, were rarely observed in nonsuckled glands. Alveolar structure was evident in nonsuckled glands, but the number of cells per alveolar cross-section was reduced (30 vs 22). Unlike in suckled glands, there was a marked gradation in classification of epithelial cells across zones in involuting glands. For example, nearly 10% of the epithelium was well-differentiated in the tissue from the upper zone, whereas no well-differentiated cells were found in the lower zones. Regression of the mammary parenchyma does not occur uniformly through the udder, so use of single biopsy to study involution should be avoided. Presence of alveoli after 42 d indicates that redevelopment of the udder with subsequent lactations is less dramatic than suggested from study of other species.     

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.     

Applicability of a progesterone-based timed artificilal insemination protocol after follicular fluid aspiration using the ovum pick-up technique in suckled beef cows

We conducted a progesterone-based timed AI protocol after follicular fluid aspiration using the ovum pick-up (OPU) technique to examine its applicability to the suckled beef cow. A total of 19 beef cows were randomly allocated to one of the following three groups based on the number of days postpartum: 13 to 60 days (Group A: suckled; early postpartum period, n=9), 61 to 150 days (Group B: suckled; mid postpartum period, n=6), or 151 to 281 days (Group C: non-suckled; prolonged open period, n=4) postpartum. These cows were treated with follicular fluid aspiration and insertion of a progesterone-releasing intravaginal device (PRID) on day 0. The PRID was removed and 500 microg of cloprostenol was intramuscularly administered on day 7. A dose (100 microg) of fertirelin acetate was injected intramuscularly 48 hours later, and this was followed by a timed AI (TAI) after another 18 hours (day 10). Serum samples were taken on days 0, 7, 9, 10, 12, 17, 24 and 31 for determination of the estradiol-17beta (E(2)) and progesterone concentrations. Pregnancy diagnosis was made by rectal palpation approximately 60 days after TAI. There was no significant difference in the peripheral E(2) concentrations among the three groups during the period of the hormonal treatment. The average progesterone concentrations in Group A on day 17 were significantly higher than those in Group B and exceeded 1.0 ng/ml on day 17 and thereafter. There was no significant difference in the numbers of collected immature oocytes among the three groups. The pregnancy rates in Groups A, B, and C were 77.8% (7/9), 83.3% (5/6) and 50.0% (2/4), respectively. In conclusion, this timed AI protocol is applicable to suckled beef cows within the period of 60 days postpartum.     

Mammary gland growth as influenced by litter size in lactating sows: impact on lysine requirement

Twenty-eight primiparous sows were used to determine the effect of litter size on the growth of mammary glands and nursing pigs during lactation. Litter size was set to 6, 7, 8, 9, 10, 11, or 12 pigs by cross-fostering immediately after birth. Four sows were allotted to each litter-size group. Sows were allowed to consume a daily maximum of 13.6 Mcal ME and 46.3 g of lysine during lactation. Sows were slaughtered on d 21 (20.6+/-1.1) of lactation. Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was separated, weighed, and ground for chemical analysis. Dry matter, dry fat-free tissue (DFFT), crude protein, ash, and DNA contents were measured. Only glands known to have been nursed were included in the data set. Wet and dry weights and the amounts of DFFT, protein, DNA, ash, and fat in individual nursed mammary glands linearly decreased (P<.05) as litter size increased. Percentages of DFFT, protein, and DNA were quadratically affected (P<.05) by litter size on d 21 of lactation. Total mammary wet and dry weights and total DFFT, protein, DNA, fat, and ash amount of all nursed mammary glands of each sow were increased as litter size increased (P<.05). Changing litter size from 6 to 12 pigs resulted in 2,098, 432, 253, 227, 4.4, 178, and 20 g increases in the amounts of total mammary wet weight, dry weight, DFFT, protein, DNA, fat, and ash, respectively, on d 21 of lactation. Litter weight gain was 18.1 kg greater in sows with 12 pigs than in sows with 6 pigs. Sows with a larger litter size had a greater increase in total mass of mammary gland tissue and litter weight but had lower growth of individual nursed mammary glands and individual pigs than sows with the smaller litter size. The need for nutrients to support additional mammary gland and litter growth as litter size increases should be considered when estimating nutrient requirements for lactating sows. Sows need an additional .96 g lysine per day to account for mammary gland growth for each pig added to a litter.     

Presence of subepithelial lymphoid nodules in the teat of ewes

A total of 87 clinically healthy ovine teats were examined bacteriologically (by scraping the mucosa) and histologically. Teats examined were those of lactating mammary glands with no bacteria isolated (n = 23); of mammary glands after cessation of lactation with no bacteria isolated (n = 25); of lactating mammary glands with bacteria isolated (n = 22); and of mammary glands after cessation of lactation with bacteria isolated (n = 17). The salient histological feature was subepithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Subepithelial lymphoid nodules, some with germinal centres, were detected in 43 (49%) teats. The majority of lymphoid nodules was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with the presence of leucocytic activity (P < 0.001) and with the presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced subepithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion. The findings call for further investigations into the lymphoid structures of the teat; these should elucidate the role and development of mammary mucosa-associated lymphoid tissues and may lead to strategies for enhancing non-specific defence mechanisms of the mammary gland.