Estradiol benzoate (0.02 mg/kg of body weight) was injected (i.m.) daily to 13 ovariectomized cows in their first or third lactations during early (60-90 days), mid (140-200 days) and late (240-300 days) lactation. The majority of the udder quarters were free of bacteria throughout the experiment. Signs of clinical mastitis were observed after 9-19 days of treatment in cows at mid and late lactation. This was accompanied by a decrease in milk yield, an increase in milk somatic cell count (MSCC) and increases in milk concentration of antitrypsin and serum albumin (as indicator of increased permeability); and milk NAGase activity (as indicator of epithelial cell damage or release of the enzyme from phagocytes). NAGase activity was first to increase and on some occasions was not accompanied by an increased MSCC. The response was either absent (first lactation) or relatively mild (third lactation) in cows at the early stages of lactation. The intensity of the inflammatory response increased with advancing lactation stage. Generally, the response of cows in the third lactation was greater than that of cows in the first lactation at a corresponding stage of lactation. Three cows which received the control treatment with vehicle alone showed no changes in any of the parameters except a slight decrease in antitrypsin. All inflammatory indicators correlated negatively with quarter milk production; milk NAGase activity was superior in this respect (r = -0.75). All correlation coefficients were generally higher after, rather than before the hormone treatment.

Bacterial growth (E. coli) in whey was studied by turbidometric technique during estradiol benzoate administration (0.02 mg/kg of body weight/day for 12-19 days) to 13 ovariectomized cows at three stages (early, mid and late) of the 1st- and 3rd-lactations. Whey samples from cows at early stage (60-90 days) of 1st-lactation promote the growth of E. coli during the estradiol treatment. This included a significant increase in the maximum turbidity and a decrease in the generation time. Bacterial growth was inhibited in whey from cows at other stages of lactation during the hormone treatment. The degree of inhibition varied at different stages of lactation. No significant alterations in the ability of whey to support bacterial growth were observed in 3 ovariectomized cows treated with the drug vehicle (arachis oil) alone. Majority of the quarters included in the present study were bacteriologically negative throughout the study.

SUMMARY Alterations in the various leukocyte populations in milk, blood, and mammary lymph were studied by use of the flow cytometric method during acute mastitis episodes induced by endotoxin infusion (50 μg of lipopolysaccharide of Salmonella typhimurium SH 4809) via the teat canal. Lymph samples were collected via a semipermanent catheter from an afferent duct to the supramammary lymph node. Milk somatic cell count increased at 4 hours after infusion of endotoxin. Neutrophils were the predominant cell population for up to 59 hours after infusion. Numbers of lymphocytes and monocytes-macrophages in milk also increased after the endotoxin infusion. The total cell count in milk started to decrease during the third postinfusion day and returned to preinfusion values during the fourth day. Lymphocyte numbers remained high for about 1 week after the infusion, and lymphocytes were the predominant cell population between postinfusion days 4 and 8. Total blood leukocyte count decreased during the first 6 hours after infusion, followed by an increase until postinfusion hour 31. The proportion of neutrophils in blood increased during the first day, whereas that of lymphocytes decreased. Lymph flow rate and leukocyte numbers in lymph increased after endotoxin infusion. The proportion of neutrophils in the lymph increased during the first 6 hours, whereas that of lymphocytes decreased. After postinfusion hour 6, the inverse course of events was seen.

The bovine blood neutrophil phagocytosis and the blood and milk lymphocyte proliferative response upon stimulation with Phytohaemagglutinin, Concanavalin A and Pokeweed mitogens was studied from 3 weeks prior to calving until 3 weeks after calving. Neutrophil phagocytosis and the total and differential blood leukocyte counts were performed by flow cytometry. A gradual increase in the percentage of phagocytized bacteria and the average number of bacteria per phagocyte was observed before calving followed by a sharp fall on the first postpartum. This was followed by a steady increase in the above parameters reaching the highest levels at two weeks postpartum. There was a gradual increase in the number of neutrophils in blood as calving approached followed by a sharp decrease after calving. The number of lymphocytes in blood dropped before calving, being at the lowest level on the day before calving. The proliferative response of blood and milk lymphocytes upon stimulation with the three mitogens was low during the week preceding parturition with the lowest value on the day before calving. The response of blood lymphocytes returned to a higher level the second week after calving while that of milk lymphocytes remained at a low level during the first and the second postpartum weeks.

SummaryOvariectomized cows were injected daily with estradiol benzoate (4–19 days). Plasma estradiol‐17 β levels under estradiol administration were comparable to the normal levels found during the last weeks of gestation. The total and differential blood leukocyte counts, milk somatic cell count (MSCC) and the phagocytic capacity of blood and milk neutrophils were determined by flow cytometry. Majority of the cows showed signs of clinical mastitis in all quarters after the treatment. Variations in this response in relation to lactation number and stage was observed. Milk yield was markedly decreased (P<0.001) and the treatment caused increases in MSCC (P<0.001) and in the percentages of blood and milk neutrophils (P<0.001) and of milk lymphocytes (P<0.01). Significant decreases in the percentages of blood lymphocytes (P<0.001) and of milk macrophages (P<0.001) were also observed. Mean percentage of phagocytized bacteria by blood neutrophils was increased (P<0.001) as also of milk neutrophils, but to a lesser extent (P<0.05). There was also an increase in the number of bacteria per phagocyte while the percentage of phagocytic cells among milk neutrophils was decreased (P<0.05). With the exception of the percentage of phagocytic cells which decreased (P<0.05), other phagocytosis parameters did not change when serum collected from cows under estradiol treatment was used in the incubation mixture.ZusammenfassungWirkungen einer exogenen Verabreichung von östrogen an ovarektomierte Kühe auf Blut‐ und Milchleukozytenzahlen sowie auf die neutrophile Phagozytose, gemessen mit einem DurchflußzytometerOvarektomierten Kühen wurden täglich östradiolbenzoat (4–19 Tage) injiziert. Bei Verabreichung von östradiol waren die östradiol‐17 β‐Werte im Plasma vergleichbar mit denen, die gewöhnlich in den letzten paar Wochen der Trächtigkeit nachweisbar sind. Die Gesamt‐ und Differentialblut‐leukozytenzahlen, die somatische Milchzellzahl (MSCC) und die Phagozytose‐Aktivität der Blut‐ und Milchneutrophilen konnten mit einem Durchflußzytometer bestimmt werden. Nach der Behandlung blieb die Mehrzahl der Kühe frei von klinischen Erscheinungen. Unterschiede in dieser Reaktion je nach Laktationsperiode bzw. Laktationsphase wurden beobachtet. Die Milchleistung nahm beträchtlich ab (P<0.001) und die Behandlung bewirkte eine Zunahme der MSCC und der Milchlymphozyten. Eine bedeutende Abnahme des Prozentsatzes sowohl der Blutlymphozyten als auch der Milchmakrophagen konnte beobachtet werden.Der Durchschnittsprozentsatz der von Blutgranulozyten phagozytierten Bakterien sowie der der Blutgranulozyten nahm zu, dieser jedoch in geringerem Maß. Eine Zunahme der Anzahl von Bakterien pro Phagozyt fand auch statt, während der Prozentsatz der phagozytierenden Zellen unter den neutrophilen Granulozyten abnahm. Mit Ausnahme des Prozentsatzes der phagozytierenden Zellen, der abnahm, blieben die anderen Parameter der Phagozytose auch dann unverändert, als Serum von Kühen, die mit östradiol behandelt wurden, der Inkubationsmischung beigemengt wurde.