Respiratory burst as a biomarker for stress responses.

A module for the detection of immunotoxic events within the test system Triple-Lux to be used during spaceflights was developed. It is based on the production of reactive oxygen species within the respiratory burst during phagocytosis or after stimulation of the phagocytes with phorbol 12-myristate 13-acetate (PMA). For this purpose, luminol-dependent chemiluminescence was measured. The assays were carried out with polymorphonuclear leukocytes purified from sheep peripheral blood. The influence of hydrocortisone and Cd2+ on the respiratory burst in polymorphonuclear leukocytes was assayed. Hydrocortisone in concentrations between 10(-4) and 10(-9) mol/liter showed an immunostimulating effect after PMA treatment. An immunosuppressive effect was observed for Cd2+ in concentrations between 10(-4) and 10(-7) mol/liter. Cryoconservation, which has often been critical for primary cells, can be accomplished without any subsequent loss of function by freezing the cells in dimethyl sulfoxide-containing medium.

Status of cryopreservation of embryos from domestic animals.

The discovery of glycerol as an effective cryoprotectant for spermatozoa led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse and later in other laboratory animals. Subsequently, these techniques were applied to domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos but no success has been reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identified as "equilibrium" cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with BSA) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. During the cooling process the straws are seeded (-4 to -7 degrees C) and cooling is continued at a rate of 0.3 to 0.5 degree C/min to -30 degrees C when bovine embryos may be plunged into LN2. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DMSO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with cooling rates and seeding and plunging temperatures similar to those used with bovine embryos. Swine embryos have shown a high sensitivity to temperature and cryoprotectants probably due to their high lipid content and a temperature decrease to 15 or 10 degrees C causes a dramatic increase in the percentage of degenerated embryos. However, a recent study has shown that hatched pig blastocysts survived exposure below 15 degrees C. Recent research has shown that embryos may also be frozen by a "nonequilibrium" method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated vitrification media (3.5 to 4.0 M) and a very rapid freeze that avoids the formation of ice allowing the solution to change from a liquid to a glassy state. Vitrification solutions consist of combinations of sucrose, glycerol, and propylene glycol. With this technique, 50% pregnancy rates have been reported with the bovine blastocyst.

Effects of oestradiol cypionate on spontaneous and oxytocin-stimulated postpartum myometrial activity in the cow.

The effects of oestradiol cypionate (ECP) on spontaneous and oxytocin-induced postpartum myometrial activity were measured in four cows using strain gauge transducers (SGT). On the first day after parturition, prior to treatment with ECP, myometrial activity consisted mainly of single-peak contractions (mean frequency 9.6/h, mean duration 141.0s, the majority of the contractions being propagated in a tubocervical direction. Injection of ECP (5 mg i.m.) 18 h after parturition led to suppression of coordinated myometrial activity and the development of sustained low amplitude contractions of reduced frequency (mean 2.9/h, P less than 0.01) and increased duration (mean 422.2 s, P less than 0.05), with multiple superimposed small peaks. In addition, all parts of the uterus tended to contract simultaneously. These changes were apparent by 4 h after treatment and persisted until day 5 after parturition. Injection of oxytocin (25 USP units i.v.) at 24 h after parturition stimulated the reappearance of single-peak coordinated contractions. However, pretreatment with ECP did not enhance the myometrial response to oxytocin.

Fertility of cows challenged with a cytopathic strain of Bovine Viral Diarrhea virus during an outbreak of spontaneous infection with a noncytopathic strain.

During an attempt to accumulate 40 Bovine Viral Diarrhea virus (BVDV) seronegative cows for breeding and for intramuscular infection on Day 21 of gestation, a persistently infected cow was inadvertently included among the first group of seronegative animals assembled. This animal proceeded to infect all seronegative animals added to the experimental herd. Since the addition of cows was gradual and they were bred as they arrived, a group of cows was bred before they seroconverted, another group was inseminated during seroconversion and a third group was seropositive when bred. First service conception rates were 22.2, 44.4 and 78.6%, respectively. The difference between 22.2 and 78.6% conception rates was significant (P < 0.05). Thirty cows were diagnosed pregnant at 21 d after service on the basis of nonreturn to estrus, presence of a palpable corpus luteum and high serum progesterone concentration. Seventeen of these received cytopathic BVDV intramuscularly and 13 cows served as controls. All control cows and 9 of 17 (52.9%) virus-treated cows had normal fetuses and placentas at slaughter on Day 70. Six pregnancies were lost between 23 and 33 d after insemination and two were lost between 35 and 40 d after insemination. Noncytopathic BVDV was demonstrated in all eight of these cows either in the buffy coat or in tissues, despite the presence of serum neutralizing antibodies.

Myometrial activity during natural and dexamethasone-induced parturition in the cow.

Myometrial activity was monitored during natural and dexamethasone-induced parturition in 8 Holstein dairy cattle, using strain gauge transducers. Four gauges were attached to the serosal surface of the gravid uterine horn, dividing it into thirds. Parturition was induced in 2 of 4 heifers and 2 of 4 cows (group 1); the remaining animals were allowed to calve spontaneously (group 2). Chains of low-amplitude contractions (repeated small deviations from base line) were detected before parturition was induced, and these were more common at distended parts of the uterus. Uncharacteristically sharp peaks followed by small rhythmic contractions, during preinduction recording, indicated that the myometrium was responsive to fetal movement even several days before parturition. By 18 hours before parturition, discrete single contractions appeared independently of contraction chains, and the first tubocervical peristaltic contraction waves were detected. The mean area under recorded contraction curves (uterine work) increased quadratically and the frequency of contractions decreased linearly from 12 hours before parturition to 2 hours after parturition. There was also an increase in the proportion of tubocervical waves over this period, and contraction chains were no longer present. During the second stage of labor, distended and undistended parts of the uterus were equally active, and forceful maternal straining was associated with larger sustained contractions. Fetal membrane rupture was accompanied by a doubling in the rate of passage of contraction waves along the length of the uterus. After the calf was expelled, contractions became extremely regular, and the majority progressed in a tubocervical direction.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature of early reproductive failure caused by bovine viral diarrhea virus.

A 2-part study was undertaken to determine the effect of bovine viral diarrhea (BVD) virus on fertilization and early development of embryos. In experiment 1, 10 seronegative cows were superovulated and artificially inseminated twice during estrus. After the second insemination, 5 of the cows received intrauterine infusion of BVD virus suspension. The other 5 cows received suspending medium only and served as controls. All 10 cows were slaughtered on day 3, and ova and embryos were collected for morphologic evaluation. A total of 49 and 52 ova and embryos were collected from the control and virus-treated cows, respectively. Among the ova and embryos collected from control cows, 81.6% were fertilized, whereas only 52% were fertilized in the virus-treated group. The statistically significant difference (P less than 0.01) indicated that the virus interferes with fertilization. In experiment 2, the protocol was identical except for slaughter on day 13. Seventy-nine ova and embryos were collected from the 6 control cows, and the 6 virus-treated cows yielded 59 ova and embryos. Of the total ova and embryos recovered on day 13, 88.6% and 50.8% were hatched and developing normally in the control and virus-treated groups, respectively. The difference was highly significant (P less than 0.001). Unfertilized ova and degenerating embryos could not be differentiated on the basis of morphologic appearance. The nearly identical percentages of unfertilized ova in experiment 1 and unhatched ova and embryos in experiment 2 strongly suggested that fertilization failure is the principal manifestation of the observed adverse effect of BVD virus infection.

Effects of exogenous follicle-stimulating hormone and luteinizing hormone in bitches.

Ten mixed-breed bitches of unknown ages, weighing 10 to 15 kg, were given a series of subcutaneous injections of follicle-stimulating hormone according to 2 different schedules, for total doses of 35 or 50 mg. Forty-eight hours after the last injection of follicle-stimulating hormone, each bitch was given 15 mg of luteinizing hormone, intravenously. The internal genitalia of 8 of the 10 bitches were recovered 24 hours after the luteinizing hormone injection and were examined histologically. External signs of estrus developed in 7 of 10 bitches in the 2 groups, indicating that estrogen was being secreted by the ovaries. Histologic examination of the ovaries revealed that many follicles had grown to the antral stage, but none had developed to the preovulatory stage, and all were atretic. Ovulation did not occur in any bitch. Endometrial proliferation and vaginal cornification that occurred in 7 of the 10 bitches were further evidence of estrogen production by the ovaries.