Assessment of neutrophil migration, phagocytosis and bactericidal capacity in neonatal foals.

Comparison of neutrophil function was made between 8 clinically normal pony foals (3 to 7 days of age), and their dams. Random migration, stimulated migration to zymosan-activated serum, bacterial phagocytosis and bactericidal capacity of neutrophils were determined in vitro. Random migration was greater (P less than 0.01) and stimulated migration was less (P less than 0.01) in foals than in their dams. Bacterial phagocytosis and bactericidal capacity of neutrophils were not different (P greater than 0.05) between foals and mares. Results of this study suggested that neonatal foals have altered neutrophil locomotion, when compared to their dams.

Effect of estradiol and progesterone on antistaphylococcal activity of neutrophils from ovariectomized mares.

Neutrophils isolated from jugular blood of ovariectomized mares were studied for the effect of estradiol and progesterone on bactericidal activity against Staphylococcus aureus. In experiment 1, neutrophils obtained from 4 mares were tested for bactericidal activity by adding estradiol (43 pg/ml) or progesterone (6.4 ng/ml) to the bactericidal assay. In experiment 2, 3 of the 4 ovariectomized mares were given 2 mg of estradiol, IM, daily for 3 days. Eighteen days after the initial estradiol injection, mares were given 300 mg of progesterone, IM, for 6 days. Neutrophils from these mares were tested for bactericidal activity 4 days after the initial estradiol injection, 17 days after the initial estradiol injection (control), and 7 days after the first progesterone injection. Bactericidal activity was measured at 30 and 120 minutes by counting the number of colony-forming units remaining. Neutrophil antistaphylococcal activity was not altered by adding estradiol and progesterone to the assay or by supplementing ovariectomized mares with estradiol and progesterone (P greater than 0.05).

Endometrial concentrations of ampicillin in mares after intrauterine infusion of the drug.

Serum concentration of ampicillin, a semisynthetic penicillin, was measured in mares at various time intervals up to 24 hours after intrauterine infusion of 3 g of ampicillin. Blood samples were drawn immediately before infusion and at 1-, 4-, 10- and 24-hour intervals after infusion. At postinfusion hour 24, two endometrial biopsy specimens were obtained to measure endometrial concentrations of ampicillin. Blood was drawn twice as part of the 24-hour postinfusion sample collection, once before removal of the biopsy specimens and again 5 minutes after removal of the biopsy specimens. After drug infusion, more diestrous mares had detectable serum ampicillin concentration than did estrous mares for all samples, except the 24-hour prebiopsy sample. None of the 24-hour prebiopsy serum samples had detectable ampicillin concentration, but ampicillin was detected in the serum of 4 of 5 diestrous mares after endometrial biopsy. Endometrial concentrations of ampicillin were detectable at postinfusion hour 24 in estrous and diestrous mares, but were not different. All 24-hour biopsy specimens had ampicillin concentrations greater than the ampicillin minimal inhibitory concentration.

Comparison of uterine protein content and distribution of bacteria in the reproductive tract of mares after intrauterine inoculation of Haemophilus equigenitalis or Pseudomonas aeruginosa.

Two groups of 3 mares were inoculated with Haemophilus equigenitalis or Pseudomonas aeruginosa on the 1st day of estrus. Uterine flushing samples were recovered on day 3 of estrus and day 8 after ovulation for each cycle. Mares were killed 22, 25, and 30 days after inoculation with P aeruginosa and 45, 46, and 49 days after inoculation with H equigenitalis. Pseudomonas aeruginosa was recovered from the uterus of 2 mares 48 hours after inoculation. Although the initial flushing sample of 1 of these 2 mares had an increased total protein concentration, there appeared to be little difference between protein concentrations of other uterine flushing samples. Haemophilus equigenitalis was recovered from the uterus of each of the 3 mares at postmortem. One mare had a slight, purulent discharge from the vulva. Total protein values were not increased in flushing samples from this mare after inoculation with H equigenitalis. Total protein values decreased in the last flushing sample of each of the 2 remaining mares. Swabbing the uterus was more effective than was homogenizing the uterine mucosa in isolating H equigenitalis.

Photomicrographic evaluation of stallion spermatozoal motility characteristics.

A photomicrographic method for evaluation of stallion spermatozoal motility was developed, and spermatozoal image and velocity characteristics were defined. The photomicrographic method was compared with visual estimation of motility in the same semen sample over time. Using photomicrography, velocities and percentages of individual spermatozoal image characteristics were determined. Although there was a high correlation between results of the 2 methods, results of the photomicrographic method were more repeatable than were those of the visual method.

Effect of bovine seminal plasma on neutrophil phagocytosis of bull spermatozoa.

Seminal plasma was obtained from bulls of known fertility and was assessed for its effect on serum-induced phagocytosis of bull spermatozoa. A non-dialysable component was found to inhibit neutrophil phagocytic uptake of spermatozoa. The component was not destroyed by heating (56 degrees C for 30 min) or removed by ether. Use of a bactericidal assay confirmed the inhibition and suggested that inhibition does not permanently impair neutrophil function. Immunoperoxidase staining demonstrated the presence of bovine IgM, IgG1 and IgG2 on spermatozoa incubated in serum. Affinity of spermatozoa for the immunoglobulins was reduced when seminal plasma was added to the serum. These results suggest that bull seminal plasma can regulate phagocytic ingestion of spermatozoa. While the mechanism of this regulation remains obscure, it may be important in providing protection to spermatozoa immediately after ejaculation.

Effect of stage of cycle, sampling frequency and recovery of micro-organisms on total protein content of mare uterine flushings.

Mares with sound reproductive tracts were divided into two groups. In Group I (N = 12), uteri were flushed once per oestrous cycle during alternate cycles while in Group II (N = 8) mares were flushed twice in a cycle for 2 contiguous cycles. Total protein concentrations and total recoverable protein of uterine flushings taken on Day 3 of oestrus and Day 8 after ovulation in each of the 2 groups and between the 2 groups did not differ significantly. The length of oestrus and dioestrus was not affected by the flushing procedures. Total recoverable protein and total protein concentrations of flushings were higher at Day 3 of oestrus and Day 8 and 15 after ovulation (P less than 0.01) when a micro-organism was isolated from the uterus before flushing.

Antibacterial activity of mare uterine fluid.

Luminal fluid from the mare uterus was used to investigate its relation to antibacterial defenses. Uterine flushings were collected at Day 3 of estrus, Day 8 postovulation and Day 15 postovulation. Uterine proteins were concentrated by ultrafiltration, dialyzed and examined for chemotactic activity to neutrophils and for antibacterial properties. Serum taken at the time of flushing was dialyzed and studied in a similar manner. Neutrophil migration in response to serum from Day 3 estrus and Day 8 postovulation was increased (P less than 0.05) above controls. Uterine protein from Day 8 postovulation and from Day 3 of estrus also stimulated neutrophil migration (P less than 0.05) above values of controls. Antibacterial activity was measured by incubation of S. zooepidemicus with concentrated uterine flushing or serum. Serum from all three estrous cycle intervals diluted 1:10 or used at a protein concentration equal to the protein concentration of uterine fluid did not inhibit growth. After 4 h of incubation, bacterial growth in estrous serum was significantly greater (P less than 0.01) than serum taken at Day 8 and Day 15 postovulation. Uterine flushings from Day 8 postovulation significantly decreased bacterial colony-forming units (P less than 0.01). Heating flushings at 56 degrees C for 30 min did not abolish the antimicrobial activity, while heating flushings for 30 min at 80 degrees C removed this activity. The antibacterial activity does not appear to be due to agglutinating antibody.

The effects of corticosteroid administration on the migration, phagocytosis and bactericidal capacity of equine neutrophils.

Neutrophil function was evaluated in six clinically normal adult horses, immediately before and 3-6 hours after they were given one dose of hydrocortisone sodium succinate (1 mg/kg body weight). Random migration, stimulated migration to zymosan-activated serum, bacterial phagocytosis and bactericidal capacity of neutrophils were determined in vitro. The mean indices of stimulated migration (net migration and migration ratio) were significantly greater after CS administration (net migration = 62 +/- 23 micron; migration ratio = 11.5 +/- 6.7) than before CS administration (net migration = 44 +/- 10 micron; migration ratio = 6.0 +/- 3.1; P less than 0.05). Random migration, bacterial phagocytosis and bactericidal capacity of neutrophils were unchanged by CS therapy. Results from this study suggest that the migration of equine neutrophils is influenced, but not impaired, after one dose (1 mg/kg) of hydrocortisone sodium succinate and that the latter causes no change in the ability of equine neutrophils to phagocytize and kill Staphylococcus aureus.

Evaluation of cellulose acetate/nitrate filters for the study of stallion sperm motility.

Stallion semen was diluted in a Hepes-supplemented buffer (CM) (10(6) spermatozoa/ml) and placed in the upper well of a Sykes-Moore chemotaxis chamber. Chambers were incubated in a humidified atmosphere (5% CO2 in air) at 37 degrees C for 1 and 2 h and spermatozoa were allowed to swim through filters with a mean pore size of 3,5 or 8 micron. Spermatozoa entered filters of all three pore sizes. Distance travelled was greater for each increase in pore size (P less than 0.01) but did not differ (P greater than 0.05) between 1 and 2h of incubation. Extended semen from stallions of different fertility was analysed for the minimal concentration of spermatozoa needed to enter filters with a 3 micron pore size. Sperm progression into the filter reflected the motility of the ejaculate. Assuming that sperm motility is a good indicator of fertility, this method may be useful for estimating the fertility of a stallion. Progression of spermatozoa into filters with a pore size of 3 micron was hampered by supernatants from overnight cultures of Streptococcus zooepidemicus and Enterobacter aerogenes. Motility decreased after 2h of incubation in supernatant from S. zooepidemicus diluted 1:5 and E. aerogenes supernatant diluted 1:5 and 1:10 in culture medium. In contrast, the bacterial supernatants were chemokinetic to horse neutrophils and did not affect their viability.

Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare.

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).