Clinical and ultrasonographic findings in the mammary gland of sheep

AIM: To compare clinical and ultrasonographic findings and assess the value of ultrasonography for evaluating the structure and condition of the teat and teat canal of sheep. METHODS: The mammary glands of 30 lactating ewes were examined clinically and using ultrasonography. The distance from the teat tip to the ground was measured and diagnostic images of the body of the gland, gland cistern and teat cistern were obtained using an 8.5 MHz frequency ultrasonic transducer. A California mastitis test (CMT) and bacteriological culture were performed on milk samples collected from each half of each udder. RESULTS: Udder parenchyma and teats were palpably normal in 27/30 (90%) ewes. Milk clots or abnormalities were evident in 4/30 (13%) udders, lesions in the glandular parenchyma and/or teats were evident by palpation in 3/30 (10%), and pathogenic bacteria were cultured from 2/30 (7%). In both of the latter cases, mastitis was diagnosed based on clotted and discoloured appearance of milk and highly positive CMT reactions. Ultrasonographic examination in these udders revealed non-homogenous regions in the glandular tissue and alternating areas of hyperechogenic and hypoechogenic tissue. Overall, highly positive CMT reactions were evident in milk from 14/30 (47%) left halves and 9/30 (30%) right halves (p=0.02). No relationship between teat-to-ground distances and CMT results was evident (p>0.05). Teat canal lengths ranged from 5.7-10.3 mm (mean=8.6; SD=1.3 mm) and the widths from 1.8-3.1 mm (mean=2.3; SD=0.4 mm). Milk sampled from ewes that had long (> or = 9 mm) or wide (> or = 2 mm) teat canals was highly positive to the CMT in 75% and 83% of cases, respectively. CONCLUSIONS: Ultrasonographic measurements of the length and width of the teat canal appeared to correlate with CMT results. The use of ultrasonography in evaluating the health of the udder of small ruminants holds promise for the future.     

Clinical and ultrasonographic findings in the mammary gland of sheep

AIM: To compare clinical and ultrasonographic findings and assess the value of ultrasonography for evaluating the structure and condition of the teat and teat canal of sheep.

METHODS: The mammary glands of 30 lactating ewes were examined clinically and using ultrasonography. The distance from the teat tip to the ground was measured and diagnostic images of the body of the gland, gland cistern and teat cistern were obtained using an 8.5 MHz frequency ultrasonic transducer. A California mastitis test (CMT) and bacteriological culture were performed on milk samples collected from each half of each udder.

RESULTS: Udder parenchyma and teats were palpably normal in 27/30 (90%) ewes. Milk clots or abnormalities were evident in 4/30 (13%) udders, lesions in the glandular parenchyma and/or teats were evident by palpation in 3/30 (10%), and pathogenic bacteria were cultured from 2/30 (7%). In both of the latter cases, mastitis was diagnosed based on clotted and discoloured appearance of milk and highly positive CMT reactions. Ultrasonographic examination in these udders revealed non-homogenous regions in the glandular tissue and alternating areas of hyperechogenic and hypoechogenic tissue. Overall, highly positive CMT reactions were evident in milk from 14/30 (47%) left halves and 9/30 (30%) right halves (p=0.02). No relationship between teat-to-ground distances and CMT results was evident (p>0.05). Teat canal lengths ranged from 5.7–10.3 mm (mean=8.6; SD=1.3 mm) and the widths from 1.8–3.1 mm (mean=2.3; SD=0.4 mm). Milk sampled from ewes that had long (≥9 mm) or wide (≥2 mm) teat canals was highly positive to the CMT in 75% and 83% of cases, respectively.

CONCLUSIONS: Ultrasonographic measurements of the length and width of the teat canal appeared to correlate with CMT results. The use of ultrasonography in evaluating the health of the udder of small ruminants holds promise for the future.     

Immunoglobulins, lysozyme and lactoferrin in the teat and udder of the dry cow during endotoxin-induced inflammation.

Immunoglobulins (Ig) and antibacterial proteins like lysozyme and lactoferrin are components of the humoral defence against infections. Changes in Ig, lysozyme and lactoferrin concentrations during endotoxin-induced inflammation in the test cistern and udder quarter of the dry cow were studied. Surgical closure of the passage between teat and udder cisterns enabled studies of reactions in the teat cistern without interference of the mammary gland. After endotoxin infusion, IgG1, IgG2, lysozyme, and to some extent IgM, increased in the teats and udder quarters, and were positively correlated with changes in somatic cell counts. No significant changes were observed in IgA or lactoferrin. The origin and significance of Ig, lysozyme and lactoferrin in the bovine teat and udder are discussed. Ig probably originated both from serum and from local plasma cells, while leukocytes appeared to be the source of lysozyme during inflammation. Secretory epithelium appeared to be the source of lactoferrin. Support for this theory was the almost total absence of lactoferrin in teat cistern samples.     

Invasive teat surgery in dairy cattle: part I - surgical procedures and classification of lesions

A prospective study was performed to identify the nature and management of teat abnormalities in cows presented to a referral teaching hospital during a three year period. All cattle (n = 60) admitted to the Ontario Veterinary College for teat problems were evaluated by physical examination; in 53 teats, contrast radiography or xeroradiography were obtained. Surgery was performed on 52 teats from 51 cows and a prosthesis was implanted in 27 teats. Short term (under two weeks) complications included intraoperative bleeding (n = 6), milk leakage through the incision (n = 4), and failure to milk by machine in 26 cases. Histopathological diagnosis of sections taken from obstructive lesions included fibrous tissue (n = 8), normal mammary tissue (n = 3), fibropapilloma, mammary polyps, and inflamed mucosa (one each). The lesion could be classified into five types: 1) focal teat cistern obstruction, 2) diffuse teat cistern obstruction, 3) membranous obstruction, 4) diffuse teat and gland cistern obstruction, or 5) leakage of milk through an abnormal route (i.e. teat fistula, webbed teat, or lacerations).     

Ultrasonographic appearance of edema caused by injections in the mammary gland attachments of dairy cows

OBJECTIVE: To determine the ultrasonographic appearance and detectability of edema induced by SC injection of mild silver protein suspension in the mammary gland attachments of dairy cows. DESIGN: Prospective study. ANIMALS: 6 lactating cows. PROCEDURE: In each cow, the number of quarters that received injections was randomly assigned. A mild silver protein susoension was injected SC into cranial and caudal mammary gland attachment sites. The number of injections and volume injected were determined on the basis of the appearance of the mammary gland and the desired subjective visual effect. Seventeen sites were chosen for injection and 7 sites did not receive injections. Ultrasonographic images were obtained 1 day prior and 6 days after injections were started. Cows received injections 1, 3, and 5 days after initial sonography. The sonographer was unaware of which sites received injections. RESULTS: Ultrasonography revealed alternating hypoechoic and hyperechoic bands at injection sites. Certain injections caused the intimal surface of the subcutaneous abdominal vein to develop a corrugated appearance. All injection sites were correctly identified ultrasonographically (100% sensitivity, 100% specificity) with a positive and negative predictive value of 1.0. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that mild silver protein suspension injected SC to enhance the appearance of the mammary glands of dairy cows can be readily detected by ultrasonography. Detection of injection sites should be made on the basis of the distribution and ultrasonographic appearance of edema.     

Teat tip reconstruction by supernumerary teat autotransplantation in cattle

OBJECTIVE: Evaluation of use of a supernumerary teat as a pedicle graft to repair experimental teat tip damage in cattle. STUDY DESIGN: Experimental study. ANIMALS: Mature, non-gravid hybrid cows with > or =1 supernumerary teat. METHODS: The main teat and adjacent supernumerary teat were examined for patency by ultrasound with a 8 MHz linear array transducer. The tip of the main teat was resected at the junction of cistern and canal. The supernumerary teat was partially resected semi-circumferentially at the cranial aspect of the base to maintain vascular supply and transplanted by end-to-end anastomosis. The pedicle of the supernumerary teat graft was amputated in stages starting at 14 days, again at 16 days and separated at 18 days. Reconstructed teat patency was evaluated at intervals by ultrasonography and confirmed at 50 days by histopathology. Repeated measures ANOVA was used to compare the measurements of luminal diameter between days; significance was set at P<.05. RESULTS: All reconstructed teats were patent. Decreased luminal diameters were detected at 25, 32, 39, and 46 days by ultrasonography. On histopathology, granulation and epithelial (mucosal) proliferation contributed to luminal reduction. CONCLUSIONS: In this experimental model of an acute damage to a teat tip, supernumerary teat grafting was successfully used to restore teat anatomy. CLINICAL RELEVANCE: Supernumerary teat pedicle grafting may be considered as a salvage technique for reconstruction of a damaged bovine teat in cows that have an adjacent patent supernumerary teat. Function of the reconstructed teat in a lactating cow remains unknown.     

B-mode ultrasonography of the bovine udder and teat

The udders and teats of cows were examined by brightness mode (B-mode) ultrasonography. A 5-MHz linear array transducer and a 5-MHz or 10-MHz mechanical sector transducer were used in the examinations. In lactating animals, the teat sinus, gland sinus, and lactiferous ducts were imaged easily. The scans also allowed visualization of the layers of the teat wall. The annular fold and the folding of the mucosa at the junction of the teat sinus and the papillary duct were seen best with the 5-MHz and 10-MHz mechanical sector transducers. In one cow lacking milk flow from one quarter, a mass at the junction of the papillary duct and the teat sinus was observed ultrasonographically. Surgical removal of the mass resulted in a return of milk flow from that quarter.     

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.     

Ultrasonography of the teat canal in cows and sheep

When the isolated teat of a cow was examined with an 8.5 MHz linear array transducer in a vertical plane, the teat canal appeared as a thin, white line, bordered on each side by parallel, thick, grey-black bands. In a horizontal plane a comparable image was obtained. In a sheep, images of comparable quality were obtained with a 12 MHz transducer. Histological studies of the tissues whose removal led to the disappearance of this characteristic ultrasonographic appearance showed that it was associated with the stratified keratinised squamous epithelium with distinct papillae. The content of keratin in the stratum corneum was apparently responsible for the bright zone; the stratum lucidum was not visible, and the surrounding dark, less echoic area was associated with the stratum granulosum. Doppler echography in live animals confirmed this designation. The outer layers of the teat wall were more echogenic.     

B‐Mode and Doppler Sonography of the Mammary Glands in Dairy Goats for Mastitis Diagnosis

This study aimed to evaluate the sonographic characteristics of the udder and teats and to determine the Doppler indexes of mammary artery in healthy and undergoing subclinical and clinical mastitis goats. Thirty animals among Saanen and Alpine Brown goats were arranged in three groups, healthy goats (HG), goats with subclinical mastitis (SMG) and goats with clinical mastitis (CMG). Using the B‐mode, the sonographic characteristics (echotexture and echogenicity) and biometry (diameter and area of the udder cistern, diameter and area of the teat cistern and thickness of the teat wall) were evaluated. Using Doppler ultrasonography, the vascular indexes of the mammary artery were obtained. It was observed hyperechogenicity with solid component in the gland cistern when comparing animals with clinical mastitis and healthy mammary tissue. Regarding the echotexture of the breast tissue, there was heterogeneity in the mammary parenchyma on the three groups, for the milk, it was observed homogeneity for animals on HG and SMG and heterogeneity for animals on CMG. Grey‐scale quantitative assessment revealed increase in echogenicity (mean value) for all the structures when comparing the three groups. Biometry did not reveal statistical difference between groups, for none of the evaluated structures. Doppler examination of the mammary artery showed the decrease of end diastolic velocity and raise of pulsatility index between groups. The association of B‐mode and Doppler ultrasonography is useful for the evaluation of the udder of dairy goats with mastitis. It is a sensitive and specific method for the study of this disease. Doppler mode was unable to establish reliable criteria for diagnosis of subclinical mastitis. Moreover, the quantification of echogenicity is a useful technique for the evaluation of the milk in animals with mastitis; therefore, it is suggested that it can be used as complementary technique for the diagnosis of mastitis in goats.     

Clinical, bacteriological, cytological and pathological features of teat disorders in ewes

We describe the features of 13 cases of teat disorders in ewes, as follows: lengthy teats (one case), papilloma infection (two), staphylococcal dermatitis (one), nodules and abscesses (two), teat bites (three), stenosis (one), orf (two), compressed teat (one). Mannheimia haemolytica, Staphylococcus epidermidis, Staphylococcus simulans or Staphylococcus aureus were isolated from samples. Sub-epithelial leucocytic infiltration at the teat duct-teat cistern border, but not lymphoid nodules, was the salient histological finding in the teat. Leucocytic infiltration and destruction of parenchyma were evident in the mammary gland. The results provide field evidence suggesting that teat lesions predispose ewes to mastitis.     

The effects of inoculation of Mannheimia haemolytica into the teat of lactating ewes

The objectives of the work described in this paper were: (i) to study the outcome of challenging ewes with Mannheimia haemolytica, at different sites of their teats, (ii) to compare the effects of two different isolates of the organism and (iii) to describe the features of the resulting lesions. Thirty-two ewes were used in the study and allocated into one of two groups (A or B, n = 16); they were challenged with one of two isolates of M. haemolytica, respectively, strain ES26L of known pathogenicity or strain VSM08L from the teat duct of a healthy ewe. Each group was further divided into four equal subgroups: the ewes in the A1/B1 subgroups were intramammarily challenged; one teat of the ewes in the A2/B2 subgroups was immersed into a broth-culture of the organisms; one teat of the ewes in the A3/B3 subgroups was inoculated 2 mm-deep, whilst one teat of the ewes in the A4/B4 subgroups was inoculated 6 mm-deep. The animals were monitored clinically, bacteriologically and cytologically before and after challenge; one animal in each subgroup was euthanised 2, 4, 7 and 11 days after challenge. All ewes in the A1/B1 subgroups developed clinical mastitis, whilst of the other animals, only one ewe in each of the A4/B4 subgroups did. Neither of the two strains used was associated with more positive bacteriological or CMT results; the A2/B2 subgroups were associated with less positive results than the A3/B3 and A4/B4 subgroups. In some ewes of the A2/B2 subgroups, mild leucocytic infiltration in the teat was evident; in the ewes of the A3/B3 subgroups, leucocytic infiltration (neutrophils, lymphocytes, plasma cells) was seen, as well as a lymphoid hyperplasia at the border between the teat duct and teat cistern; in ewes of the A4/B4 subgroups, intense subepithelial leucocytic infiltration was the salient feature. No differences were found in the severity of lesions between the two strains used or the three treatments carried out. Although strain VSM08L had been isolated from the teat duct of a healthy ewe, it caused mastitis when inoculated intramammarily; although strain ES26L is of known pathogenicity for the mammary gland, it did not cause clinical mastitis when deposited 2 mm-deep into the teat. These findings point to a protective role of the teat of ewes, which appear to limit bacterial penetration from the teat duct or cistern to the mammary gland. The lymphoid tissue, at the border between the teat duct--teat cistern, may play a significant protective role.     

Postoperative evaluation of the surgical treatment of accessory teat and gland cistern complexes in dairy cows.

The purpose of this study was the long-term evaluation of a method of surgically repairing the abnormal condition of accessory teat and gland cistern complexes in dairy cattle. A prospective evaluation of three cows that had undergone the procedure was done. These cows were evaluated from four months to one year, postoperatively. A thorough history, physical examination, contrast radiography, and ultrasonic examination were done on each cow. A retrospective evaluation of an additional 13 cows that had also undergone the procedure was obtained four months to three years following the surgery, via owner communication. The prospective portion of this study demonstrated patency of the communication between main and accessory teat cisterns. Postoperative complications included initial slow milking and mild swelling of the involved teat. These problems resolved in all cows one to two months postoperatively. None of the 16 cows that had undergone the surgery either developed or was treated for mastitis. Likewise, none of these cows was sold or slaughtered as a result of postoperative teat problems or unsatisfactory milk production. Our study demonstrated that this particular method of surgical correction of accessory teat and gland cistern complexes is effective, is esthetically acceptable, produces minimal associated complications, and preserves the milk production capacity of the gland.     

Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression

Staphylococcus aureus is a prolific mastitis-causing bacterium that resides naturally in the environment of the dairy cow. The aim of this study was to profile immune gene expression in tissue from the alveolar, ductal, gland cistern and teat canal regions of the bovine mammary gland following intramammary infection with S. aureus. Quantitative real-time PCR (qPCR) was used to profile expression of innate immune genes including pattern recognition receptors (PRRs), cytokines, antimicrobial peptides (AMPs) and acute phase proteins (APPs). Consistent expression of Toll-like receptors (TLRs) 1-10 and NOD-like receptors (NODs) 1-2 was detected in all four tissue regions. Pro-inflammatory cytokines (IL6, IL17A and IL8) and anti-inflammatory cytokine (IL10) were induced in all 4 tissues. APP (SAA3 and HP) and AMP (DEFB4 and DEFB5) genes showed the greatest induction throughout the mammary gland in response to S. aureus, with particularly high expression in alveolar tissue (SAA3 and HP >133- and >80-fold respectively, P<0.05; DEFB4 and DEFB5 >9- and >27-fold respectively, P<0.05). Collectively, our data show both sentinel and effector immune functions throughout the mammary gland in response to S. aureus challenge.     

Evaluation of the Microbiological Status of Milk and Various Structures in Mammary Glands from Naturally Infected Dairy Cows

A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections. The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter. A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination. Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples. When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%). There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antibody-containing cells and specialised epithelial cells in the bovine teat

Tissue from the ends of teats of dry, periparturient and lactating cows were studied using light and electron microscopy. Accumulations of infiltrating leucocytes mainly in the folds of the distal rosette of the teat cistern (Furstenberg's rosette) were detected; plasma cells predominated. The latter were classified by the type of immunoglobulin (Ig) which they synthesised. Plasma cells synthesising IgG1 were found to be the major antibody producing cell type of the teat. Neither the number of stromal plasma cells present nor the class of Ig which they synthesised was significantly altered by changes in mammary gland secretory activity. Scanning electron microscopy revealed areas of epithelium of Furstenberg's rosette that contained cells differing in surface characteristics from epithelial cells of adjacent areas of the teat cistern.     

Tuberculin elicited cellular immune response in the lactating bovine mammary gland vaccinated intramammarily with Mycobacterium bovis

The development of a local antigen-specific sensitivity was monitored histologically and in secretions of the bovine mammary gland. Three cows in mid-lactation were immunized by injecting 50 microliter of a killed Mycobacterium bovis vaccine into the dorsal secretory tissue of the rear mammary glands; two cows served as unvaccinated controls. Ten weeks after vaccination, all cows were challenged by intramammary infusion of 1.0 microgram tuberculin in 5 ml phosphate-buffered saline (PBS). Three quarters of each cow received tuberculin at 72, 48, and 24 hours before slaughter; a control quarter received PBS at 72 hours. Vaccinated cows exhibited an intense, local cellular reaction to tuberculin in teat-end tissues at all times post infusion; PBS-infused glands were normal. A moderate leukocyte response in parenchymal tissues adjacent to the gland cistern of tuberculin-infused quarters was observed, but deep parenchymal tissues were normal; no effect on milk yield was found. Tuberculin-infused quarters exhibited histological responses in teat cisternal tissues similar to those in delayed-type hypersensitivity reactions. Leukocytic accumulation was primarily macrophages and lymphocytes with few neutrophils. Erythema was observed in the distal half of the cistern, and fibrin deposits were found in subepithelial connective tissues. The epithelium, although distended with leukocytes, was intact and numerous mitotic figures were present. Unvaccinated cows showed no response to challenge. Results demonstrated a marked and specific cellular response in sensitized cows to challenge with tuberculin.     

Epidemiology of mammary gland disorders in multiparous Finnish Ayrshire cows

Logistic regression was used to investigate the effects of host characteristics, milk production and 23 veterinary diagnoses on the odds of contracting acute and chronic mastitis, and teat injury among 41 989 multiparous Finnish Ayrshire cows that calved during 1983.

Cows which had higher previous-lactation milk yields were at increased risk of acute and chronic mastitis, and teat injury. All of the mammary gland disorders were directly interrelated. Abomasal disorder, indoor hypomagnesemia, prolapsed uterus and vagina, and abortion were not risk factors for any of the three mammary gland disorders; however, retained placenta, udder edema, ketosis, non-parturient paresis, rumen acidosis, traumatic reticuloperitonitis, silent heat, ovarian cyst and other types of infertility were all direct risk factors for at least two mammary gland disorders.     

A study of the development of endotoxin-induced inflammation in the bovine teat.

Endotoxin-induced local inflammation was studied by frequent samplings in a bovine teat cistern model, which provides a unique possibility for in vivo studies of reactions in the teat without interference from the mammary gland. A rapid inflammatory response of rather short duration was elicited after endotoxin administration. An initial increase in the concentrations of bovine serum albumin and N-acetyl-beta-D-glucosaminidase, indicating a disturbance in the epithelial integrity, was observed between 1 and 1.5 h post infusion (p.i.). Approximately 0.5 h later, the first influx of leukocytes, mainly neutrophils, appeared. The neutrophils tended to enter the teat cistern in several peaks occurring between 2.5 and 5 h p.i. The sampling procedure decreased the accumulation of cells by approximately 40%, which was probably due to the removal of inflammatory mediators at an early stage. The parallel use of 2 teats instead of 1 had no major influence on the inflammatory process. This teat cistern model and the experimental procedure used should be suitable for further studies of the development of local inflammation.     

Effect of liner design,pulsator setting,and vacuum level on bovine teat tissue changes and milking characteristics as measured by ultrasonography

: Friesian-type dairy cows were milked with different machine settings to determine the effect of these settings on teat tissue reaction and on milking characteristics. Three teat-cup liner designs were used with varying upper barrel dimensions (wide-bore WB = 31.6 mm; narrow-bore NB = 21.0 mm; narrow-bore NB1 = 25.0 mm). These liners were tested with alternate and simultaneous pulsation patterns, pulsator ratios (60:40 and 67:33) and three system vacuum levels (40, 44 and 50 kPa). Teat tissue was measured using ultrasonography, before milking and directly after milking. The measurements recorded were teat canal length (TCL), teat diameter (TD), cistern diameter (CD) and teat wall thickness (TWT).Teat tissue changes were similar with a system vacuum level of either 50 kPa (mid-level) or 40 kPa (low-level). Widening the liner upper barrel bore dimension from 21.0 mm (P < 0.01) or 25.0 mm (P < 0.001) to 31.6 mm increased the magnitude of changes in TD and TWT after machine milking. Milk yield per cow was significantly (P < 0.05) higher and cluster-on time was reduced (P < 0.01) with the WB cluster as compared to the NB1 cluster. Minimum changes in teat tissue parameters were achieved with system vacuum level of 40 kPa and 50 kPa using NB and WB clusters, respectively. Similar changes in teat tissue and milk yield per cow were observed with alternate and simultaneous pulsation patterns. Widening pulsator ratio from 60:40 to 67:33 did not have negative effects on changes in teat tissue and had a positive effect on milk yield and milking time. Milk liner design had a bigger effect on teat tissue changes and milking characteristics than pulsation settings.