Surface plasmon resonance investigations of human epidermal growth factor receptor 2.

This investigation utilizes surface plasmon resonance (SPR) spectroscopy to detect and quantify human epidermal growth factor receptor 2 (HER-2), an oncogene product that is over-expressed in some aggressive forms of breast cancer. Specifically, the HER-2 trans-membrane protein p185 and its extra cellular fragment p105 are analytes targeted in this work by using a gold-based biosensor slide on which an anti-HER-2 antibody has been immobilized by attachment to Protein G that is fixed to the gold film. A detection limit of > or =11 ng/mL for p185 resulted when trastuzumab was used as the anti-HER-2 antibody on the biosensor slide. Experiments with semi-purified p105 revealed that it binds weakly and reversibly to trastuzumab, therefore complicating its detection and quantification. Results of studies that reacted a 13-amino-acid peptide (PP13) from the HER-2 kinase domain with its specific antibody were critically different than p185 and p105 studies. Spectral analysis of the reflectivity at constant bulk buffer refractive index revealed a progressive negative SPR shift over time. A negative shift suggests that a loss of protein mass from the anti-PP13 antibody-Protein G biosensor is occurring. Several possibilities that may explain these negative SPR shifts are discussed.

Zidovudine therapy is associated with an increased capacity of phytohemagglutinin-stimulated cells to express interleukin-2 receptors. Pittsburgh AIDS Clinical Trial Unit.

Zidovudine therapy of AIDS patients has been shown to cause only transient improvements in the numbers of circulating CD4+ cells and the in vitro functional activities of cultured lymphocytes. The present studies were undertaken to determine whether prolonged zidovudine therapy enhanced reactivity in two sensitive assays of T-cell function: the ability of phytohemagglutinin (PHA)-stimulated cells to form T-cell colonies and their capacity to express receptors for the growth factor interleukin-2 (IL-2). Treated patients, studied over periods of 20-60 weeks, showed no improvement in colony formation at any time interval, even in plates supplemented with exogenous IL-2. However, mitogen-stimulated T lymphocytes showed a significant increase in the capacity to express IL-2 receptors (CD25). This enhanced expression resulted primarily from activation of the CD8+ cell subset.

Some effects of intrauterine devices on reproductive funtion in the ewe.

PIP: This presentation is limited to the antifertility effect of the IUD in ewes and to some of the changes in uterine function caused by its presence. Some of these changes may occur only in sheep. A custom-made plastic spiral, 5 cm long and 5-8 mm outside diameter, was inserted into the lumen of only 1 uterine horn. Ovum fertility occurred normally in control ewes but the fertility rate of IUD-wearing ewes was very low, as determined by ovum cleavage at 3 days after breeding. None of the 17 ova was cleaved when ovulation occurred on the spiral side and only 1 in 10 was cleaved when ovulation occurred opposite the spiral. Accessory sperm were not found in the zona pellucidae of any ovum in IUD-carrying ewes while in controls accessory sperm ranged from 10 to over 60. These findings indicate the IUD did more than interfere physically with the passage of sperm up the reproductive tract. The ova was found not defective nor was ovulation interfered with. Myometrial tissue removed and tested in vitro showed adequate contractility. However an altered motility pattern in test animals was shown by in vivo observations of exteriorized uteri. This might partly account for the absence of sperm from the oviducts. Sperm cells placed into the uterine horns were found to be broken into heads and tails or to have disappeared after 5 hours. This effect was markedly greater in horns containing the IUD. Some sperm cells were observed being phagocytized by leukocytes. Spermicidal activity in excised and washed uterine horns could be observed as early as 1 hour after semen injection. This activity was largely stopped when total uteri were excised. Phagocytosis of sperm cells was increased when an inflammatory reaction was producted by bacterial inoculation. Iv dye injections showed resulting dye concentrations directly beneath the turns of the spiral (p less than .01), which was considered to indicate the degree of leakage from the endometrial vascular system. Other experiments showed an IUD intensifies bacterial capabilities of the uterus by increasing the inflammatory response to the presence of foreign substances, whether semen or bacteria. It was concluded that as IUD in the sheep uterus inhibits sperm transport and ovum fertilization, perhaps by changing the nature of uterine motility.^ieng

Comparative fertility of normal and repeat-breeding cows as embryo recipients.

Morphologically normal embryos were transferred surgically into uteri of normal and repeat-breeder cows at seven days post-estrus to compare embryo survival rates in the two kinds of cows. All cows were less than ten years of age and had no abnormal genital discharges, cystic ovarian follicles, or anatomical abnormalities of the reproductive tract. Normal cows had not been inseminated after last calving. Repeat-breeders had at least four infertile services within the past six months (average of 6.2 services after calving). To test fertility of repeat-breeders at synchronized estrus, 22 anatomically-normal repeat-breeders were treated by intramuscular (i.m.) injection with prostaglandin F(2)alpha (PGF(2)alpha) on day 11 of an estrous cycle (estrus = day 0) and inseminated at induced estrus; 11 cows (50%) had a normal fetus at necropsy on day 60. Twenty-three repeat-breeders and 23 normal cows were assigned as embryo recipients and treated i.m. with PGF(2)alpha to synchronize estrus. All embryo donors were normal cows. Donors were treated with FSH and PGF(2)alpha and inseminated at estrus. On day 7 after estrus, embryos were recovered nonsurgically from donors and one embryo was transferred through a flank incision to the anterior end of the uterine horn adjacent to the corpus luteum of each recipient. Recipients that did not return to estrus were necropsied at day 60. Of 28 normal and 23 repeat-breeder recipients, 23 normal cows (82%) and 16 repeat breeders (70%) were pregnant at day 60 (P=0.235). Thus, at seven days post-estrus, the maternal environment of most of these repeat-breeders was satisfactory for maintaining pregnancy.

Inhibition of sperm transport and fertilization in superovulating ewes.

In Experiment 1, all ewes were treated with follicle stimulating hormone (FSH-P) to induce superovulation. Ewes came into natural estrus or were treated with prostaglandin F(2)alpha (PGF(2)alpha) or 6-methyl-17-acetoxyprogesterone (MAP) to regulate the time of estrus. The ewes were mated during estrus and necropsied 3 h after mating. Regulation of estrus with either compound reduced the number of sperm recovered from the cervix, uterus, and oviducts and increased the proportions of sperm recovered from the cervix and uterine body that were immotile, dead, or had disrupted membranes. In Experiment 2, all ewes were in natural estrus. They either ovulated naturally or were superovulated, and ewes in each group were necropsied at 3 or 23 h after mating. Superovulation reduced the number of sperm in oviducts, uterus, and anterior segments of the cervix at both time intervals and increased the proportions of sperm that were immotile, dead, or had disrupted membranes. In Experiment 3, of 3x2 design, ewes were in either natural estrus or estrus regulated with PGF(2)alpha or with MAP; they ovulated naturally or were superovulated. Ewes were necropsied 3 d after mating and ova were examined. Both regulation of estrus and superovulation reduced the proportion of ova that were fertilized and reduced the number of accessory sperm attached to fertilized ova.

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts.

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but uninjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.

Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm.

Three experiments were conducted with 105 superovulating Holstein dairy cows in attempts to improve the fertilization rate. Cows were superovulated with follicle-stimulating hormone (FSH) and time of estrus was regulated with prostaglandin F(2)alpha (PGF(2)alpha). Semen was deposited on each infundibulum through a laparoscope inserted through the flank (Experiment 1) or near the uterotubal junctions through flexible tubing passed through the cervix and uterine horns (Experiment 2). In the third experiment, high numbers of sperm in fresh semen were deposited in the uterus. Cows were necropsied and ova were recovered and examined about 3.5 d after the beginning of estrus. Deposition of 0.5 ml of frozen-thawed semen on each infundibulum (Experiment 1) reduced both ovum recovery and fertilization. In ten cows inseminated on the infundibulum, ova representing 43% of ovulation points were recovered and 9% of these recovered ova were fertilized. In ten control cows, ova representing 80% of ovulation points were recovered and 62% of them were fertilized. In a 2 x 2 experiment with 36 superovulating cows (Experiment 2), 1 ml of diluted fresh or frozen semen was deposited either near the uterotubal junction or in the uterine body. The overall fertilization rate was 61%, with no significant effect of site of semen deposition or type of semen used. In Experiment 3, 2 or 3 ml of neat semen (average of 4.4 billion sperm) was deposited in the uterus of 12 cows; 183 of 197 intact ova (93%) were fertilized. In 56 control cows inseminated with 0.5 to 1.5 ml of frozen diluted semen (average of 70 million sperm), 502 of 947 intact ova were fertilized (53%, P<0.001). Insemination with high numbers of fresh sperm overcame problems of sperm loss or sperm transport and improved the fertilization rate.

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes.

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.

Progestagen induced sperm breakage in the sheep vagina.

PIP: The author, working for the U.S. Department of Agriculture at Beltsville, Maryland, researched sperm breakage in the vaginas of parous ewes inserted with progestogen-impregnated intravaginal sponges, which regulate ovulation. The cylindrical sponges were 2 cm in diameter and 8 cm long. Some sponges were impregnated with 60 mg of medroxyprogesterone acetate (MPA). Ewes were artificially inseminated. Sperm cell breakage was significantly greater (p less than .01) in the MPA-sponge ewes than in the blank-sponge (5) and control (untreated, 5) ewes. It was clearly seen that the increase in sperm breakage in the MPA-sponge ewes resulted from the MPA and was unrelated to the sponge itself. The same increased breakage was seen when the MPA sponge was removed before the corpus luteum regressed. Whether the MPA sponge was left in place for 1 or 2 weeks had no effect.^ieng

Effect of unilateral cornual insemination upon fertilization rate in superovulating and single-ovulating cattle.

In Exp. 1, 21 first-service cattle and seven repeat-breeder cattle, averaging 4.7 infertile services, were brought into estrus and superovulated by treatment with follicle-stimulating hormone and prostaglandin F2 alpha. At insemination, semen was deposited in the greater curvature of one uterine horn, about midway between the utero-cervical junction and the utero-tubal junction. Cattle were necropsied 2 to 7 d after estrus and ova were recovered and examined. The fertilization rate for first-service cows was 74% of 362 intact ova and for repeat-breeders, 43% of 128 intact ova (P less than .001). Fertilization rate in first-service cows was 81% on the side of semen deposition and 68% on the opposite side (P less than .01); the rates in repeat-breeders were 54% and 32% (P less than .025). Differences between sides were due mostly to four cows that averaged 93% fertilization on the side of semen deposition and 19% on the opposite side. The proportion of fertilized ova with accessory sperm (17%) did not differ between sides of the reproductive tract. In Exp. 2, 60 first-service and 32 repeat-breeder cows in natural estrus had semen deposited in the uterine body or in the greater curvature of one uterine horn, either on the side of impending ovulation or on the opposite side. At necropsy, 55 ova were recovered from first-service cows, of which 42 (76%) were intact and 13 (24%) were ruptured or fragmented. Of the 42 intact ova, 41 (98%) were cleaved. From the 32 repeat-breeders, 30 ova were recovered, of which 26 (87%) were intact and 4 (13%) were ruptured; 23 of the 26 intact ova (88%) were cleaved. Site of semen deposition had no significant effect on either fertilization rate or number of accessory sperm in either type of cow. First-service cows averaged more accessory sperm (40) than did repeat-breeders (19, P less than .01). Overall results indicated that sperm deposited deep in one uterine horn fertilized ova nearly as frequently in the opposite oviduct as in the adjacent oviduct except in 14% of superovulating cattle.

Inhibition by progesterone of IUD-induced luteal regression in the ewe.

PIP: A U.S. Department of Agriculture study at Beltsville, Maryland investigated the effect of progesterone on IUD-induced luteal regression in 95 ewes. The IUD was a plastic spiral with diameter 9 mm and length 30 mm. The first experiment suggested that IUD-induced regression of a corpus luteum (CL) is dependent upon the stage of the estrous cycle but unrelated to the age of the CL. When ovulation was induced on Cycle Day 5 in 4 ewes and an IUD was inserted on Day 7, neither the original CLs, about 6 days old, nor the induced CLs, about 1 day old, regressed. Experiment 2 provided evidence that exogenous progesterone generally prevents IUD-induced luteal regression. 29 ewes were injected twice daily on Days 1-4 of a cycle with 10 mg of progesterone in corn oil or with corn oil alone. An IUD was inserted on Day 3. Sacrifice was on Day 9. Early CL (ovaries ipsilateral to IUD) regression occurred in 7 in the corn-oil group but in only 2 in the progesterone group. In Experiment 3 the administration of progesterone to estradiol- and IUD-treated ewes tended to reduce the percentage of ewes with regressed CL's (p=.10, ovaries ipsilateral to IUD). Estradiol itself extends the stage during which the insertion of an IUD causes luteolysis.^ieng

Attempts to overcome the anti-fertility effect of intrauterine plastic spirals in the ewe.

PIP: A study was conducted to determine whether the antifertilization of intrauterine devices in ewes could be overcome by the use of hormones or drugs that either stimulate or inhibit uterine motility. 44 parous, western ewes were unilaterally ovariectomized, and a plastic spiral was inserted into the uterine horn opposite the remaining ovary. Spirals were immediately removed from 5 ewes. At the time of mating, 2 weeks to 3 months post surgery, sham-operated and spiral control ewes were injected with saline solution. Ewes with spirals were injected with either estradiol, epinephrine, oxytocin, oxytocin and acetylcholine, or oxytocin, acetylcholine, and physostigmine. Other ewes with spirals were laparotomized, and either the uterus was massaged or the cervical lumen was cleaned. 3 days postmating, 8 ova recovered from 5 sham-operated ewes were all cleaved, with a mean of 90 accessory sperm in the zonae pellucidae. 3 of 12 ova recovered from 8 spiral control ewes were cleaved. Only 1 of 40 ova recovered from 28 treated ewes was cleaved. Accessory sperm were not found in either cleaved or uncleaved ova from any spiral ewe. None of the treatments overcame the antifertilization effect of the spiral.^ieng

Effect of prostaglandin F2 alpha, phenylephrine and ergonovine on uterine contractions in the ewe.

Two experiments were conducted to determine the effect of prostaglandin F2 alpha (PGF2 alpha), phenylephrine and ergonovine on uterine contractions. In the first experiment, ewes were bilaterally ovariectomized, and a strain gauge force transducer was sutured to the serosa of one uterine horn. Each ewe was treated sc with 2 micrograms of estradiol-17 beta daily to prevent regression of the uterus. Beginning at least 5 d after ovariectomy, four dose levels of PGF2 alpha, phenylephrine and methoxamine were given by im injection and ergonovine was given by im or iv injection. Phenylephrine, methoxamine and ergonovine are alpha-adrenoceptor agonists. Uterine activity was recorded by physiograph for 30 min before and 90 min after treatment. Tracing were analyzed for 20-min periods before treatment and 4 to 24 min and 50 to 70 min after treatment. In Exp. 2, transducers were attached to uteri of intact ewes at d 10 to 12 of an estrous cycle. During subsequent estrus, one or two dose levels of PGF2 alpha, phenylephrine and ergonovine were given by im injection and uterine activity recorded. In Exp. 1, PGF2 alpha and phenylephrine increased (P less than .05 or .01) the number of amplitude of contractions at both 4 to 24 and 50 to 70 min. Ergonovine given im increased the number of contractions. In intact estrous ewes, PGF2 alpha increased the number and amplitude of contractions at 4 to 24 min, phenylephrine increased the number and amplitude at both 4 to 24 and 50 to 70 min, and ergonovine increased the number slightly but significantly at 4 to 24 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Improvement by ergonovine of sperm transport, fertilization and pregnancy rates in ewes in natural or prostaglandin-induced estrus.

Eight experiments were conducted with 451 ewes to test effects of ergonovine, prostaglandin F2 alpha (PGF2 alpha) and phenylephrine on sperm transport and fertility. In most experiments, ewes were mated at estrus and necropsied 2 or 3 h later. Sperm were flushed from the oviducts, uterus and anterior, middle and posterior thirds of the cervix and counted. Various doses of PGF2 alpha or phenylephrine given im at mating caused no significant increase in sperm numbers in any segment of the tract 2 h later. Three different dose levels of ergonovine were given im to ewes in natural estrus 1 h after mating and ewes were necropsied 3 h after mating. Doses of .2 and 1.0 mg were ineffective, but .5 mg increased sperm numbers about 10-fold in the oviducts and uterus. When given im at the time of artificial insemination, .6 mg of ergonovine increased the fertilization rate at 3 d from 5/25 in control ewes to 12/25 (P less than .05). In three experiments with ewes in PGF2 alpha-induced estrus, .6 mg of ergonovine increased sperm numbers in the cervix and uterus at 3 h after mating and in the uterus and oviducts at 23 h, near ovulation. Other ewes were artificially inseminated in the external cervical os and one-half of the ewes were given .6 mg of ergonovine im; ewes not returning to estrus were laparotomized at 22 to 26 d and embryos removed. After insemination during natural estrus with .2 ml of semen, pregnancy rates were 14/25 for control ewes and 15/25 for ergonovine-treated ewes; after insemination during natural estrus with .1 ml of semen, 6/35 and 18/35 (P less than .005); after insemination during PGF2 alpha-induced estrus with .2 ml of semen, 7/60 and 12/60. Fertilization and pregnancy rates combined were 32/145 (22%) for all control ewes and 57/145 (39%) for ergonovine-treated ewes (P less than .005).

Effect of gonadotropin-releasing hormone on estrus, ovulation, and ovum cleavage rates of dairy cows.

In a 2 x 2 factorial experiment, 110 Holstein cows (55 first-breeding and 55 repeat-breeding), free of genital abnormalities, were injected i.m. at the time of insemination with either saline solution or 100 micrograms of GnRH. Blood samples were drawn from the tail vein or artery of 22 cows (10 first- and 12 repeat-breeding) immediately before GnRH injection and again 1 and 2 h later to determine whether the GnRH induced the release of LH. The GnRH caused LH release in 18 of 22 cows, with a greater (P < .05) mean concentration of LH than that in saline-treated cows at 1 h (2.3 vs 7.0 ng/mL) and 2 h (2.5 vs 6.0 ng/mL) after injection. Length of estrus and time of ovulation were calculated from estrus checks and ovarian palpations per rectum at 8-h intervals. The GnRH injections produced no change in duration of estrus (19.2 h) or time of ovulation postestrus (9.5 h). The cows were slaughtered 41 to 90 (mean = 60) h after ovulation to determine the ovum cleavage rate and the number of accessory sperm in the zona pellucida. The GnRH treatment increased the incidence of twin ovulations but did not increase the number of accessory sperm or improve the proportion of ova that cleaved in either first-service or repeat-breeding cows.

Effect of homogenized embryos on IUD-induced luteal regression in the ewe.

PIP: The effect of homogenized embryos on IUD-induced luteal regression in the ewe was studied. Plastic spiral IUDs were inserted in an uterine horn adjacent to an ovary containing a corpus luteum during various times of the estrous cycle. The ewes were sacrificed on Day 6 for examination of the corpus luteum. Estrous Cycle Day 3 was the last day in which the IUD insertion induced luteal regression. Corpus luteum was maintained in ewes when the IUD was inserted on Day 4, 5, or 6. Corpus luteum regression was not prevented when homogenized 14- or 15-day-old embryos were injected into the uterus at the time of IUD insertion. Ewes which received an IUD, a cannula, and an infusion of saline or homogenized embryos on Days 3, 4, and 5 maintained corpora lutea in 4 of 7 embryo-infused ewes and in none of 7 saline-infused ewes (p less than .05). It was determined that the IUD must remain in the uterine horn for longer than 1 day to induce corpus luteum regression and that embryos can sometimes prevent the IUD-induced regression.^ieng

Interaction of stage of the breeding season and intrauterine devices on estrous cycle length in the ewe.

PIP: To determine whether the presence of IUDs would reduce estrous-cycle length throughout a breeding season, 51 and 58 ewes, respectively, were studied in 2 experiments. The estrous cycles of ewes with an IUD adjacent to each ovary averaged 9.6 days in length from September through November, and nearly 20 days from December through February. The same ewes showed a similar pattern in a second September-to-February breeding season. In the third breeding season, ewes with an IUD in each uterine horn were laparotomized if they had not returned to heat within 9 days after a designated estrous period. Of 54 ewes laparotomized during the course of the breeding season, corpora lutea 1) had regressed or were regressing in 49 (91%) by Day 9, and 2) had regressed in the other 5 ewes by Day 18. It is concluded that cycles longer than 9 days were usually caused by 1 or more successive ovulations without estrus, and that the increased mean cycle length for IUD-bearing ewes during the second half of the breeding season was due primarily to a greater frequency of ovulation without estrus.^ieng

Increased numbers of sperm in the oviducts and improved fertilization rates in rabbits after administration of phenylephrine or ergonovine near the time of insemination.

Phenylephrine, an alpha-adrenoceptor agonist, was administered im to does near the time of mating or insemination. The treatment increased sperm numbers in the oviducts by about 50-fold and in the uterus by about 10-fold at 2 or 2.5 h after insemination. Methoxamine, another alpha-adrenoceptor agonist that was given im, did not increase sperm numbers, although both phenylephrine' and methoxamine significantly increased the number and amplitude of uterine contractions when contractions were measured by strain gauge force transducers attached to the uterus of conscious does. Ergonovine, an ergot derivative given im, increased sperm numbers more than 10-fold in the oviducts and five to 10-fold in the uterus at 2 or 2.5 h after insemination. Ergonovine increased the frequency and amplitude of uterine contractions when given iv but not when given im. In tests with a range of doses of phenylephrine and ergonovine, 5 mg of phenylephrine and .6 mg of ergonovine appeared to be near optimal for maximizing the number of sperm in the uterus and oviducts at 2.5 h after insemination. Phenoxybenzamine, an alpha-adrenoceptor blocking agent, prevented the phenylephrine-induced increases in both uterine contractions and sperm numbers in the oviducts and uterus. Phenoxybenzamine also prevented the effect of ergonovine on sperm numbers. In does inseminated with low numbers of sperm (92,000; an inseminate selected to result in a low fertilization rate in control does), the administration of phenylephrine or ergonovine significantly increased ovum fertilization rates (16% for control does, 52 and 63%, respectively, for phenylephrine- and ergonovine-treated does).

Effect of acetylcholine, prostaglandin F2 alpha and estradiol on number of sperm in the reproductive tract of inseminated rabbits.

Acetylcholine, injected im into does immediately after natural mating, significantly increased the number of sperm recovered 1 h later from the vagina and cervices but not from the oviducts. Acetylcholine caused immediate increases in the frequency and amplitude of uterine contractions as measured by strain-gauge force transducers attached to the serosa and recorded by physiograph; contraction patterns returned to the preinjection pattern within 30 min. Administration of prostaglandin F2 alpha (.75 mg) at the time of insemination increased sperm numbers significantly at 2.5 h in the oviducts, uterus and cervices; .15 or 3.75 mg caused relatively little increase in sperm numbers. Prostaglandin F2 alpha increased the number and strength of uterine contractions for more than 30 min. Estradiol injected 1 h before artificial insemination increased the number of sperm recovered from the oviducts, uterus, cervices and vagina at 2.5 h after insemination. Estradiol caused little if any detectable increase in the frequency or strength of uterine contractions during the period of increased retention and transport of sperm. When does were inseminated with low numbers of sperm (86,000), prostaglandin F2 alpha and phenylephrine, used for comparison, each increased the ovum fertilization rate (22% for control does; 76 and 73%, respectively, for prostaglandin F2 alpha- or phenylephrine-treated does).

Effect of liver microsome enzyme-inducing drugs on blood serum pentobarbital clearance and sleep time in sheep and rabbits.

Sheep were administered phenobarbital, diphenylhydantoin, chlorcyclizine, or phenylbutazone over 27 days. Rabbits were administered phenobarbital, diphenylhydantoin, or chlorcyclizine for 15 days. After the last treatment, pentobarbital was administered intravenously, and measurements were made of sleep time and blood serum pentobarbital concentrations over a 2-hour period. Treatment of ewes with phenobarbital or phenylbutazone increased the rate of pentobarbital clearance from the circulation and shortened sleep time; treatment with diphenylhydantoin or chlorcyclizine decreased the rates of pentobarbital clearance from blood and increased sleep time. Treatment of rabbits with phenobarbital or diphenylhydantoin accelerated pentobarbital clearance from blood and shortened sleep time; chlorcyclizine had no effect on blood pentobarbital concentrations or sleep time. The results suggest that accelerated or delayed clearance of pentobarbital from the circulation was largely responsible for shortened or prolonged sleep times. Other factors may have been involved to some extent in determining sleep time, because ewes with shortened sleep time tended to awake with lower circulating concentrations of pentobarbital than did control ewes, and those with prolonged sleep time tended to awake with higher pentobarbital values.