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.

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.

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)

Estrus-related odors in milk detected by trained dogs.

Dogs trained to identify vaginal mucus samples and other fluids from estrous cows were used for further studies of estrus-related odors in milk. In the first experiment, milk was collected from 10 cows during estrus and luteal phase of the estrous cycle and tested in trials of 9 samples each; 1 or 2 of the samples were from estrous cows. Dogs responded to milk from estrous cows in 76% of the exposures to such samples with significant differences among cows and significant improvement by the dogs during the experiment. Responses to milk from luteal-phase cows, averaged 6%. In a second experiment, milk was frozen and thawed four times without decreasing the frequency of response to samples from estrous cows. In the third and fourth experiments, milk was collected from four cows on several days of the estrous cycle. After the dogs were trained to respond to milk from pre-estrous cows (1 or 2 days before estrus), they responded to 64% of these samples; after the dogs were retrained to not respond to milk from pre-estrous cows, they responded 21% of the time to milk obtained 1 to 3 days before estrus. In these two experiments, responses to milk from estrous cows averaged 83% and to milk from luteal-phase cows, 8%. Trained dogs can distinguish milk of pre-estrous cows from milk of either estrous or luteal-phase cows.

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).

Intensification by glucocorticoids of induced acute leukocytic responses in the rabbit uterus.

Sexually mature does were given cortisone acetate, hydrocortisone acetate, or 9 alpha-fluoroprednisolone by intramuscular injection for 3 days, then the uterine lumens were inoculated with Escherichia coli. The glucocorticoids doubled the number of leukocytes that migrated into the uterine lumen during the acute inflammatory response. The corticoids had similar effects in estrous, ovariectomized, and pseudopregnant rabbits. The administration of hydrocortisone acetate nearly doubled the number of polymorphonuclear (PMN) leukocytes in the circulation within 24 hours. The high numbers of circulating PMN leukocytes were maintained over the 3 days of treatment. The increased leukocytic response to induced uterine infection was similar in magnitude to the increase in numbers of circulating PMN leukocytes; the corticoid-induced neutrophilia was probably responsible for the intensified leukocytic response to uterine infection.