Genetic engineering of livestock.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

Inhibin suppresses human chorionic gonadotropin secretion in term, but not first trimester, placenta.

Inhibin is produced by the placenta, with serum concentrations rising throughout pregnancy. In contrast, hCG serum concentrations peak in the first trimester and are 80-90% lower at term. This study was designed to determine the effect of inhibin on hCG secretion both early and late in gestation. Villus tissue from 3 term and 3 first trimester (8-10 week) placentas was maintained in an in vitro explant culture model for 5 days. Tissue from each placenta was incubated with control medium in 24 replicate wells for the first 72 h. During the final 48 h, 12 wells received control medium, and 12 wells received medium containing 1% rabbit antiserum raised against the alpha-subunit (residues 1-32) of the human inhibin peptide. The antiserum demonstrated biological activity by increasing serum FSH concentrations in an immature female rat bioassay. The relative increase in hCG secretion at the conclusion of days 4 and 5 in control and antiserum-treated groups for each first trimester and term placenta were compared to pretreatment hCG concentrations on day 3. The relative increases in hCG secretion of first trimester control groups on day 4 (mean +/- SD, 34 +/- 11%) and day 5 (63 +/- 23%) were compared to those in antiserum-treated groups on day 4 (39 +/- 13%) and day 5 (54 +/- 5%) and showed no significant difference between groups on either day. The same comparison in term cultures showed the relative increases in hCG secretion of control groups on day 4 (31 +/- 10%) and day 5 (64 +/- 50%) to be significantly lower than those in antiserum-treated groups on day 4 (100 +/- 41%) and day 5 (150 +/- 108%; P less than 0.001). These findings suggest that inhibin suppresses hCG secretion in term, but not first trimester, placentas.

Immunoaffinity chromatography of bovine FSH using monoclonal antibodies.

Bovine FSH (bFSH) was used to immunize BALB/c mice. Spleen cells were fused to the SP 2/0 cell line to produce hybridomas that secreted monoclonal antibodies to bFSH. One of these antibodies (USDA-bFSH-MC28) was extensively characterized and found to be a gamma 1 with kappa light chains, having extremely low cross-reactivity with other bovine pituitary hormones and with ovine and porcine FSH. The dissociation constant as measured by Scatchard analysis was 4.3 nmol/l, and proved to be in a very useful range for affinity chromatography. In an essentially one-step immunoaffinity chromatography procedure, bFSH was easily isolated in a single chromatographic step from crude anterior pituitary homogenate with better yield and with the same purity as classical chromatographic techniques.

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Starting to add worse: effects of learning to multiply on children's addition.

A major stumbling block in acquiring a new skill can be integrating it with old but related knowledge. Learning multiplication is a case in point, because it involves integrating new relations with previously acquired arithmetic knowledge (in particular, addition). Two studies explored developmental changes in the relations between single-digit addition and multiplication. In the first study, third-graders, fifth-graders, and adults performed simple addition or multiplication in mixed- and blocked-operations formats. Substantial interfering effects from related knowledge were found at all age levels, but were more pronounced for younger subjects. Thus in the early stages of learning multiplication, one consequence of learning a new operation is interference in performance of an earlier, related, but less recently studied skill. Consideration of error patterns supported the view that the problem of integrating operations is a prominent one even in the early stages of mastering multiplication. Patterns of errors were generally consistent across all age groups, and all groups were much more likely to give a correct multiplication response to an addition problem than the reverse. A second, longitudinal study confirmed this finding, showing evidence for impaired performance of addition over time within individual children (second-, third-, and fourth-graders) tested on simple addition and multiplication over a 5-month period. Analysis of reaction times for addition indicated that second-graders in advanced math classes and third-graders in regular math classes tended to slow down over the year in responses to addition problems. Fourth-graders, on the other hand, tended to increase their speed of addition over the course of the year. Multiplication showed a different pattern during this period, with no evidence for slowing among children who were able to perform this task. Disruption of previously learned knowledge in the course of acquiring new skills provides evidence that new knowledge and old knowledge are being integrated. This kind of non-monotonic development may provide an empirical method for determining the functional limits of a domain of knowledge.

In vitro development and implantation rates of fresh and cryopreserved sibling zygotes.

OBJECTIVE: To compare the developmental potential of cryopreserved human zygotes after thawing to sibling zygotes that were transferred without cryopreservation. METHODS: Retrospective analysis of embryo data and pregnancy outcome for all in vitro fertilization (IVF) patients who had sufficient zygotes to allow fresh embryo transfer and cryopreservation of additional sibling zygotes for later use. RESULTS: Zygotes survived cryopreservation at a high rate (87%). After thawing, cryopreserved zygotes developed at rates similar to those of fresh zygotes. Pregnancy occurred at similar rates after replacement of fresh embryos (27.9%) or replacement of cryopreserved-thawed zygote-derived embryos (24.3%). CONCLUSIONS: Human embryos can be cryopreserved at the pronuclear zygote stage with little loss of developmental potential. Cryopreservation allowed a reduction in the number of embryos transferred during an IVF retrieval cycle, thereby reducing the occurrence of multiple pregnancy. The total cumulative pregnancy rate per retrieval cycle doubled when pregnancies from cryopreserved-thawed zygotes were added to those originating from fresh zygotes.

Follicle size and implantation of embryos from in vitro fertilization.

OBJECTIVE: To determine the effect of increased follicle size at the time of hCG administration on the outcome of in vitro fertilization (IVF). METHODS: Fifty women immediately before 50 immediately after a protocol change were reviewed retrospectively. The criterion for hCG administration was changed from a leading follicle size of at least 17 mm to one of at least 20 mm (average of two dimensions). Changes in follicle size and number, oocytes recovered, fertilization rate, embryo development, implantation rate, and pregnancy rate were examined. Women over 40 years of age, those undergoing intracytoplasmic sperm injection, and those whose stimulation did not include GnRH agonists were excluded. RESULTS: The number of large follicles increased, but peak estradiol concentrations and number of oocytes recovered did not differ when hCG was administered at a follicular size of at least 20 mm. The numbers of oocytes fertilized, embryos replaced, and embryos cryopreserved were not statistically different. The percentage of cleaved fertilized oocytes was greater and the degree of embryo fragmentation smaller when hCG was administered at an increased follicular size. The implantation rate was higher and clinical and ongoing pregnancy rates tended to be higher when hCG was administered at a larger follicle size. CONCLUSION: The administration of hCG at a larger follicle size appears to have a beneficial effect on the outcome of IVF. Embryo quality-as reflected by decreased fragmentation, increased cleavage, and increased implantation rate-appears to be improved when hCG is delayed until two or more follicles reach at least 20 mm in diameter.

One hundred unstable bladders.

OBJECTIVE: To investigate the correlation between presenting symptoms and cystometric data in patients with urodynamically proven detrusor instability. METHODS: A retrospective review was conducted of standardized urodynamic data bases and cystometrograms from the gynecologic urodynamics laboratories at Duke University Medical Center, Durham, North Carolina, and Grady Memorial Hospital, Atlanta, Georgia. RESULTS: Of the 100 patients reviewed, 86% complained of urge incontinence and 78% complained of urinary urgency; however, 76% also complained of stress incontinence. Statistical evaluation of symptoms by analysis of variance showed no significant correlations between the symptoms of urinary frequency and urgency and any of the cystometric indices. Patients with motor urge incontinence had significantly smaller mean cystometric bladder capacities than patients without this complaint (299.9 +/- 132.5 versus 553.6 +/- 173.6 mL; P < .01). One-third (34) of the patients had mixed incontinence due to genuine stress incontinence as well as detrusor instability. Compared with patients suffering from detrusor instability alone, patients with mixed incontinence had significantly larger cystometric bladder capacities (396.0 +/- 172.9 versus 308.1 +/- 154.8 mL; P < .02) and a lower amplitude of the maximum detrusor contraction (38.8 +/- 21.7 versus 49.9 +/- 25.1 cm H2O; P < .04). CONCLUSIONS: Women with detrusor instability represent a diverse population with a wide variety of symptoms and urodynamic findings. Patients with mixed incontinence may represent a subpopulation distinct from those with pure detrusor instability. Further research that attempts to describe other discrete subpopulations of patients with detrusor instability may improve our understanding of this troublesome clinical problem.

Previous fertilization failure with conventional in vitro fertilization is associated with poor outcome of intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the outcome of intracytoplasmic sperm injection (ICSI) in patients with a history of fertilization failure with conventional IVF. DESIGN: Retrospective analysis of 2 years of clinical experience with ICSI. SETTING: Clinical IVF-ET program in a tertiary care referral center. PATIENT(S): The results of ICSI performed between January 1995 and December 1996 were compared between patients with normal semen parameters and a history of fertilization failure (< 20% of oocytes) with conventional IVF and patients with male factor infertility. INTERVENTION(S): In vitro fertilization with ICSI. MAIN OUTCOME MEASURE(S): Parameters examined included oocyte survival, fertilization, embryo cleavage, implantation rates, and clinical pregnancy rates. RESULT(S): Fertilization was achieved with ICSI for all patients during the study period. Although oocyte survival and fertilization did not differ between groups, the pregnancy and implantation rates for patients with a history of idiopathic fertilization failure (20% and 6%, respectively) were significantly lower than those for other patients undergoing ICSI (47% and 22%, respectively). CONCLUSION(S): The outcome of ICSI varied depending on the indication for treatment. Patients who had a history of failed or poor fertilization in vitro with apparently normal semen parameters had significantly lower pregnancy and implantation rates than did patients with either obstructive azoospermia or impaired semen quality.

Effect of a synthetic platelet activating factor on steroidogenesis of cultured porcine granulosa cells.

An embryo-derived platelet activating factor has been demonstrated to play an important role in reproduction. This report examined the effect of various doses of a synthetic platelet activating factor on the production of progesterone by porcine granulosa cells in culture. Granulosa cells aspirated from ovarian follicles of prepubertal gilts were grown for 24 hours in Dulbecco's Modified Eagles Media: Ham's F-12 with 5% fetal bovine serum and 1 micrograms/ml insulin. Cells were washed once in serum-free media and then cultured for an additional 48 hours with 0 to 5000 ng/ml of the platelet activating factor in media containing either 0.25% bovine serum albumin or 1% fetal bovine serum. Cells grown with fetal bovine serum produced 50% of the amount of progesterone that was produced in the absence of serum. Low doses of the platelet activating factor caused a slight decrease in progesterone production. Higher doses (greater than 500 ng/ml) in serum-free media caused a marked decrease in progesterone production. Serum had a protective effect at high doses of platelet activating factor which was probably mediated by enzymatic degradation of the platelet activating factor. In summary, platelet activating factor had no stimulatory effect on production of progesterone by porcine granulosa cells in culture.

Progress on gene transfer in farm animals.

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.

Vaccination with F-strain Mycoplasma gallisepticum to reduce production losses in layer chickens.

The effect of Mycoplasma gallisepticum (MG) infection or vaccination of Conn. F-strain MG on 45 weeks of egg production was analyzed using production records from 132 flocks of commercial layer hens. The flocks were located in Pennsylvania, and the data were collected for two years. On the average, layers maintained free from infection with MG laid 15.7 more eggs/hen housed than the MG-infected layers; figures were adjusted for layer-strain effect. This adjusted advantage decreased to 8.7 eggs/hen housed when uninfected flocks were compared with vaccinated flocks. Adjusted average production of vaccinated flocks was 7.0 eggs/hens housed more than production of MG-infected flocks. Egg production of four layer strains was observed with respect to vaccination or natural infection with MG. The four strains responded similarly whether vaccinated or infected. Route of vaccination and age of layer at time of vaccination influenced egg production of vaccinated birds. The adjusted average production/hen housed was 4.9 eggs greater for birds vaccinated via drinking water than for birds vaccinated via spray. The adjusted average was 10.3 eggs/hen housed greater for birds vaccinated between 8 and 18 weeks of age than for birds vaccinated after 18 weeks.

A simple in vivo bioassay for inhibin-like activity using ovariectomized ewes.

Long term ovariectomized ewes were used in a bioassay for inhibin-like activity. The concentration of FSH 6 to 7 hr after injection of follicular fluid (a rich source of inhibin), as a percentage of pretreatment, regressed on the log of the dose had a slope of -26.0 +/- 7.6 (5 replications, mean +/- SD) and an index of precision of .32 +/- .04. This system was rapid, relatively easy and specific for in vivo inhibin-like activity. This bioassay was also used to determine the relative potency of an affinity-purified fraction of follicular fluid.

Effect of bovine growth hormone gene expression, sex and age on plasma gonadotropins, estrone and testosterone in prepuberal pigs.

Chronic supraphysiological blood levels of growth hormone (GH) may retard sexual maturation in swine. Pigs used in this study included four founder transgenic pigs (two gilts and two boars) expressing a mouse transferrin (TF) promoter fused to a bovine (b) GH structural gene, 13 second- or third- generation transgenic pigs (seven gilts and six boars) expressing a mouse metallothionein (MT) promoter fused to a bGH structural gene and 16 control littermates (eight gilts and eight boars). Blood plasma levels of LH, FSH, estrone and testosterone were measured to determine whether expression of bGH genes altered secretion of hormones between 80 and 180 days of age. Presence of a bGH gene was detected by hybridization of DNA in dot blots of tail biopsies. Expression of a bGH gene was detected by radioimmunoassay of plasma bGH. In four TFbGH founder transgenic pigs bGH ranged from 164 to 1948 ng/ml; in one MTbGH transgenic boar of line 3104 bGH was 1211 ng/ml; and in 12 pigs of line 3706 bGH ranged from 25 to 190 ng/ml. Expression of bGH in transgenic pigs lowered (P = .0192) plasma LH with no significant differences between sexes, had no significant effect on plasma FSH and lowered plasma estrone (P = .0001) and testosterone (P = .0269) in boars (but not gilts). Plasma estrone and testosterone were higher (P = .0001) in boars than in gilts. Plasma FSH was higher (P = .0001) in gilts than boars and decreased (P = .0001) with advancing age in gilts but not in boars.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of ovariectomized ewes with bovine follicular fluid decreases secretion of FSH without changing secretion of LH.

Ovariectomized ewes were injected with 0, 0.25, 0.5, 1.0 or 2.0 ml of charcoal-extracted bovine follicular fluid. Treating ewes with 2 ml of follicular fluid resulted in a decrease in circulating concentrations of FSH to 72.8% of the pretreatment value. With smaller doses of follicular fluid, the magnitude of the decrease was less. Concentrations of LH did not change significantly. Pretreatment of ovariectomized ewes with estradiol and/or progestogen did not alter the magnitude of the FSH decrease. This action of follicular fluid extract fits the effect of the non-steroidal substance known as inhibin or folliculostatin.

Inhibition and subsequent rebound of FSH secretion following treatment with bovine follicular fluid in the ewe.

Plasma concentrations of gonadotropins were examined after treatment of ewes with bovine follicular fluid (FF) in four experiments. Mean concentrations of FSH were significantly decreased by FF treatment. The FSH depression appeared to continue throughout the length of treatment when the duration of treatment was 2-4 days. However, in an experiment in which the treatment period was 8 days, mean concentrations of FSH initially declined and then returned to control levels during the last 4 days of treatment. In all experiments, a rebound in FSH concentrations was found 24-36 h after cessation of FF treatment. The magnitude of this rebound appeared to be proportional to the degree of FSH suppression during FF treatment.

Response of plasma LH and FSH to gonadotropin-releasing hormone in pony foals and ovariectomized pony mares.

Plasma FSH and LH response to a synthetic GnRH analog was measured in adult ovariectomized pony mares (OVX) and in pony foals (<70 days of age) during late spring (May-June). FSH and LH responded in a similar fashion (200% increase) in the OVX mare, which is different from other reports for intact mares. There was a greater mean response to a comparable dose of GnRH in the prepubertal foal for both FSH (500%) and LH (900%) than in the OVX mare. There was a positive correlation between age and the maximum FSH response to GnRH in male and female foals. The LH response was positively correlated with age in male foals, but not in females. The response to GnRH in the prepubertal foals was consistent with the previously observed patterns of gonadotropin secretion during this age period.

Seasonal LH profile in ovariectomized cattle.

Two experiments were conducted to examine seasonal changes in circulating LH concentrations in ovariectomized heifers. In experiment 1, four Holstein heifers were ovariectomized in April 1977 during middiestrus. Blood samples were collected daily for 30 days surrounding each equinox and solstice for one year to examine changes in plasma LH levels at the time of seasonal photoperiod changes. The LH concentrations were highest during the winter solstice period and lowest during the summer solstice period. In addition, samples taken at two-week intervals indicated a distinct LH profile with maximal LH concentrations during November-April and minimal concentrations during May-October. In experiment 2, eight Holstein heifers were ovariectomized in June-July, 1979 and given an estradiol or a control implant in October. A distinct LH profile for the interval extending from January, 1980 to February, 1981 was found in the heifers that were not treated with estradiol. Concentrations were maximal during December-April and minimal during May-November. The LH profile followed a similar pattern in the estradiol-treated heifers; however, the overall profile was at a higher level. These data indicate that underlying seasonal reproductive mechanisms are present in cattle even though the species ovulates and breeds throughout the year.

Donor insemination. Prevalence of antibodies to cytomegalovirus.

OBJECTIVE: To determine the prevalence of seropositivity to cytomegalovirus in women presenting for donor insemination and in their partners. STUDY DESIGN: Retrospective examination of test results for patients presenting for donor insemination. Two hundred eighteen couples presenting for donor insemination were studied. RESULTS: Overall, 37% of women were seropositive for cytomegalovirus. Their partners had a slightly, although not significantly, lower rate of seropositivity. No clear relationship between patient age and rate of seropositivity was found. CONCLUSION: The rate of seropositivity to cytomegalovirus in couples presenting for donor insemination appears to be lower than that reported for the general population.

Effect of charcoal-extracted porcine follicular fluid and 17 beta-estradiol on follicular growth and plasma gonadotropins in gilts fed a progesterone agonist, altrenogest.

Thirty-four gilts in two experiments were fed altrenogest for 18 d to block spontaneous growth of ovulatory follicles after luteolysis. They were injected with estradiol or charcoal-extracted porcine follicular fluid (pFF) to determine 1) whether gonadotropin secretion could be depressed and 2) whether exposure to reduced levels of gonadotropins would result in decreased numbers of medium follicles (3 to 6 mm in diameter). Gilts in Exp. 1 received treatments in a 2 X 2 X 2 factorial arrangement starting 48 h before the last feeding of altrenogest. Corn oil or estradiol (2 micrograms/kg body weight), 5 ml of charcoal-extracted porcine serum (pS) or pFF were injected im four times at 8-h intervals and gilts were sacrificed 24 or 96 h after last feeding of altrenogest. In Exp. 2, gilts received one of four treatments consisting of 1) pS, injected iv nine times at 8-h intervals starting 48 h before the last feeding of altrenogest; 2) pFF, with injection protocol the same as for pS; 3) estradiol injected im three times and 4) four times at 8-h intervals starting 0 and 24 h, respectively, before the last feeding of altrenogest. Compared with pS or corn oil, estradiol increased (P less than .001) plasma estrogen and decreased (P less than .05) plasma luteinizing hormone (LH) without a significant effect on plasma follicle stimulating hormone (FSH). Estradiol, compared with corn oil, decreased (P less than .01) the number of medium follicles from 24.8 to 0/gilt and decreased (P less than .05) the weight of ovarian follicular fluid from 4.2 to 2.1 g/gilt at 72 h after the first injection. Five milliliters of pFF had no significant effect on plasma gonadotropins or number of medium follicles. However, 20 ml of pFF, compared with pS, decreased (P less than .05) plasma FSH from 45 ng/ml to 9 ng/ml 32 h after the first injection, had no effect on plasma LH, decreased (P less than .01) the number of medium follicles from 29.2 to 2.2/gilt and decreased (P less than .01) follicular fluid weight from 3.9 to 1.6 g/gilt by 72 h after the first injection. These results indicate that estradiol or a non-steroidal component of follicular origin can decrease secretion of gonadotropins and suppress recruitment of medium follicles in the pig.