H.W. Hawk

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milks ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Gamete transport in the superovulated cow

Abstract Reduced efficiency of sperm transport in superovulated cows can be inferred from fertilization rates, which generally average about 65%, or about 20% lower than those found in single-ovulating cattle. Also, most fertilized ova recovered from superovulated cattle have no accessory sperm in the zona pellucida, which suggests minimal numbers of sperm at the site of fertilization. Low fertility of either cows or bulls reduces the fertilization rate, probably through an effect on survival of sperm or efficiency of sperm transport in the cow. Other factors that affect fertilization rate, presumably also acting through sperm survival or transport, include age of the cow, season of the year, interval after calving, stage of the estrous cycle at which the superovulation treatment is begun, total dose of FSH used, number and timing of artificial inseminations, and site of deposition of the inseminate. Use of purified FSH to induce superovulation has improved the fertilization rate markedly in some instances, and insemination with high numbers of sperm in fresh undiluted semen has raised the fertilization rate above 90%. The apparent inhibition of sperm transport in superovulated cows may be mediated through changed patterns of steroid hormone secretion. Ova seem to pass through the oviducts faster in superovulated cows than in single-ovulating cows. The rate of passage may be shortened by a day or more in some cases, but the proportion of superovulated cows with hastened ovum transport is not known, and the consequences of rapid transport on survival of embryos has not been ascertained.

IMPROVED YIELDS OF BOVINE BLASTOCYSTS FROM IN VITRO-PRODUCED OOCYTES. I: SELECTION OF OOCYTES AND ZYGOTES

Abstract Three experiments were conducted with 7,901 oocytes (6,152 cleaved zygotes) to identify methods to increase the proportion of oocytes aspirated from ovaries of slaughtered cows that would develop to expanded blastocysts by 9 d after exposure to spermatozoa. In the first experiment, oocytes were matured for 24 h in TCM-199, fertilized, and presumptive zygotes were co-cultured in TCM-199 and granulosa cells. Cleaved zygotes from oocytes classified as good on the basis of quality of investments and cytoplasm developed to expanded blastocysts more frequently than did zygotes from intermediate-marginal oocytes (29% vs 24%; P=0.035). In the second experiment, marginal oocytes were subdivided into those with partially expanded cumulus, coarsely granulated cytoplasm or minimal cumulus (not more than 3 layers of cumulus cells, or zona pellucida partly exposed), matured in Hams F-10, and co-cultured in Menezos B2 with buffalo rat liver (BRL) cells. Cleaved zygotes from oocytes with partially expanded cumulus and/or coarsely granulated cytoplasm developed to expanded blastocysts as frequently (47%) as did good/intermediate oocytes (40%), but zygotes from oocytes with minimal cumulus developed less frequently (23%; P

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.

Improved yields of bovine blastocysts from in vitro-produced oocytes. II. Media and co-culture cells

Three experiments were conducted with 9,472 oocytes, aspirated from ovaries of slaughtered cows (7,318 cleaved zygotes), to compare some media and co-culture cells for in vitro maturation of oocytes and culture of embryos. The endpoint was development to expanded blastocysts at 9.0 d after exposure of matured oocytes to sperm. In the first experiment, oocytes were matured in TCM-199 and co-cultured in TCM-199 or Menezos B2 (B2), with granulosa or buffalo rat liver (BRL) cells. The type of co-culture cell had no effect on the proportion of oocytes or cleaved zygotes that developed to expanded blastocysts, but 31% of cleaved zygotes developed to expanded blastocysts in B2 compared with 21% in TCM-199 (P<0.001), and embryos developed faster in B2. In the second experiment, oocytes were matured in TCM-199, Hams F-10 (F-10), or B2, then all were co-cultured in B2 with BRL cells. The proportion of cleaved zygotes that developed to expanded blastocysts was 28%, 40%, and 8% after maturation in TCM-199, F-10, or B2, respectively (P<0.001). In the third experiment, presumptive zygotes were vortexed to remove cumulus at 15 hr after exposure to sperm and classified as having dense even cytoplasm or thin and/or uneven cytoplasm. Then zygotes were co-cultured with BRL or bovine oviduct epithelial (BOE) cells. There was little difference in the proportion of cleaved zygotes that developed to expanded blastocysts after co-culture with BRL (51%) or BOE cells (54%), but embryos developed somewhat faster with BOE than with BRL cells. Presumptive zygotes with even cytoplasm, compared to presumptive zygotes with uneven cytoplasm, had higher cleavage rates (85% vs 76%, P<0.001) and higher rates of development of cleaved zygotes to expanded blastocysts (59% vs 44%, P<0.001). In these experiments, the best combination for growing embryos in vitro was F-10 maturation medium, B2 embryo culture medium, and BOE cells for co-culture.

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.

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.

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.

Identification of pronuclei in in vitro fertilized cow embryos

Abstract Experiments were conducted to determine the efficiency of visualizing pronuclei in living zygotes. Oocytes aspirated from ovaries of slaughtered cows were matured for 24 hr, fertilized, and examined by differential interference contrast microscopy (DICM) to identify pronuclei, then fixed, stained and examined by phase contrast microscopy (PCM) for stage of development. In the first experiment, 1028 oocytes were examined at 3‐hr intervals between 10 and 22 hr after exposure to sperm. In the second experiment, 759 oocytes were classified by type of cumulus investment (complete, incomplete, and all others, such as loose, expanded, or no investment) and examined at 13 or 19 hours. The proportion of oocytes and embryos in which two pronuclei were seen by DICM increased from 5% at 10 hr to 38% at 19 and 22 hr, with greatest increase between 10 and 13 hours. In exp. 2, oocytes with incomplete investments had the highest proportion of zygotes with two pronuclei. Over both experiments, the proportion of e...

Death of sperm in the cervix of ewes bearing intrauterine plastic spirals or threads

Abstract A plastic spiral intrauterine device (IUD) in the ewe inhibits sperm transport through the cervix. In Exp. 1, plastic spirals were inserted surgically into the lumen of one or both uterine horns. In Exp. 2, Dacron threads were placed in the lumen of each horn. At a subsequent estrus, ewes were mated and necropsied 2 hours later. Sperm were washed from the uterine body and from the anterior, middle and posterior one-third of the cervix and examined. Plastic spirals significantly reduced the percentage of sperm recovered from the uterine body and each segment of the cervix that were motile and live and had normal acrosomes. In the anterior one-third of the cervix, e.g., 49% of the sperm were motile and 66% were live in control ewes but only 17% were motile and 23% were live in IUD ewes. Intrauterine threads also reduced the percentages of sperm that were motile and live in the anterior cervix. Failure of sperm transport in IUD-bearing ewes may be caused by conditions which result in loss of sperm motility in the cervix, thereby inhibiting the establishment of a reservoir of live sperm for transport to the oviducts.