Genetic engineering of laboratory and livestock mammals.

Recent advances in recombinant genetics have made possible the transfer of cloned genes from one organism to the genome of another. Research with mice made transgenic by insertion of rat or human genes has provided direct evidence that transferred genes can be incorporated into the germline and expressed in the recipient. Current technology for gene transfer involves microinjection of the recombinant genes into the male pronucleus of the zygote. Resulting transgenic mice, when mated as adults, produced offspring that contained and expressed the transgenes. These observations serve as indications of the possibilities that exist for genetic engineering in livestock species. Although there are some technical problems to be overcome before livestock embryos can be genetically altered by these means, the genes for producing growth hormone transgenic livestock are currently available, and research groups are working toward this objective. In addition to this work with growth hormone genes, there are many other potential applications for genetic engineering livestock to produce more highly efficient production; however, there is considerable research to be done before the full potential of this technology can be achieved. It will be necessary to identify other genes that have potential for improving the production efficiency of livestock, and it will be necessary to gain a more complete understanding of the developmental and molecular biology of livestock. The potential impact of this technology in farm animal production is enormous, but, in the short term, it will be a costly endeavor.

Effects of dichlorvos on lymphocyte reactivity during pregnancy in the pig.

This experiment was performed utilizing a total of 21 primiparous crossbred sows to determine whether a state of immunological suppression occurs during the latter stages of pregnancy in the pig and whether dichlorvos treatment affects immunological function of sows. Immunological reactivity was monitored by the use of in vitro lymphocyte transformation experiments employing the mitogens phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). The degree of blastogenesis of lymphocytes recovered from animals on this experiment was not affected by dichlorvos treatment. Blastogenesis was not affected by stage of pregnancy except in the case of that induced by LPS, which decreased (P less than .05) with advancing pregnancy. Sera from animals in this experiment were tested in a PHA-induced lymphocyte transformation experiment and it was found that sera from control animals were not inhibitory at any time. On the other hand, sera from dichlorvos-treated sows inhibited (P less than .01) PHA-induced blastogenesis. The possibility exists that dichlorvos may increase fetal survival by inhibiting the ability of the sow to immunologically damage the fetus.

Increased litter size in gilts by intrauterine infusion of seminal and sperm antigens before breeding.

Three experiments were conducted to evaluate the effect of exposure of the uterus to semen at least 3 wk before breeding on subsequent reproductive performance. In Exp. 1, uterine exposure to semen was performed three times. At least 3 wk elapsed between each treatment. Control gilts received saline infusion. All gilts were bred by artificial insemination using semen from the same boars used for semen treatment. At farrowing, significantly more (10.35 vs 8.39) pigs/litter were produced by semen-treated gilts than controls. However, due to a disease outbreak (porcine parvovirus) during the last two-thirds of the farrowing period, live pigs born and litter size at 2 wk were not statistically different between the two groups. Gilts that farrowed were rebred and no difference in their reproductive performance for second litters was noted. For Exp. 2 and 3, a single semen treatment was given using freeze-thaw killed semen at estrus before breeding. Within both Exp. 2 and 3, semen-treated gilts had a larger average litter size, but no significant differences were detected. When data for nondiseased animals in Exp. 1 and animals in Exp. 2 and 3 were pooled, it was found that total pigs born (P less than .01), live pigs born (P less than .01) and live litter birth weight (P less than .1) were all greater in semen-treated gilts than in controls. Thus, these results indicate that reproductive efficiency in gilts can be improved by intrauterine treatment with boar semen before insemination to elicit conception.

Angio-proliferative lesions in a homosexual boar with features resembling Kaposi's sarcoma.

Clinical, biopsy and necropsy studies of a homosexual boar revealed angio-proliferative lesions in the skin of the thigh, scrotum and the inguinal lymph node. Angiofibromas were identified in the dermis and subcutis of the thorax and mandible. The avidin-biotin-peroxidase complex technique demonstrated Factor VIII-related antigen as a marker for the neoplastic endothelial cells of the tumors. This boar also showed weight loss and lymphopenia. Explants of tumors were maintained in cultures for 22 passages and cultured cells produced tumors when injected subcutaneously into nude mice. No viral antigen was detected in the cultured tumor cells. Similarities and differences between the lesions in the boar and human Kaposi's sarcoma are discussed.

Effects of steroids on bovine T-lymphocyte blastogenesis in vitro.

Day 13 to 24 bovine conceptuses convert C-19 and C-21 neutral steroids to 5 beta-reduced steroids with great efficiency. Pregnancy steroids have been reported to be immunomodulatory in several species. This study examined the possibility that 5 beta-reduced products of bovine conceptus steroid metabolism, precursors, related steroids, progesterone or cortisol might affect bovine T-cell blastogenesis. The steroids tested were 5 alpha-androstan-17 beta-ol-3-one, androstene-3, 17-dione, 5 beta-pregnane-3,20-dione and 5 beta-pregnane-3 alpha,20 alpha-diol. The ability of each steroid, over a dose range of 10(-2) to 10(4) nM, to affect phytohemagglutinin (PHA)-induced or concanavalin A (Con A)-induced bovine T cell blastogenesis, or the mixed lymphocyte reaction (MLR) was evaluated. Five lactating Holstein cows served as lymphocyte donors for mitogen studies and two, that exhibited strong MLR, were donors for the MLR evaluation. Of the seven steroids tested none affected blastogenesis in a dose-related manner except for cortisol, which suppressed Con A-induced lymphocyte transformation as well as the MLR. Cortisol did not affect PHA-induced blastogenesis. Thus, of the pregnancy steroids tested at the concentrations noted, none had significant immunomodulatory effects.

Immunosuppression in the mouse by porcine uterine secretory protein.

Experiments were performed to determine whether uterine secretory proteins (PAP) of the pig affect T cell or B cell functions in vivo. T cell functions examined were E-rosette formation (ERF) and cutaneous reactivity (to phytohemagglutinin) in mature BALB/c females. Two ERF experiments of identical design were performed using five groups of ten mice each. Each mouse was presensitized with sheep erythrocytes (SRBC) designated as day 0; and the various groups were treated with saline, pig serum (PS), or PAP at different times. For cutaneous reactivity, the area of erythema at the sensitization site (pinna of ear) was measured, and three groups of 11 mice were used. These groups also received either PS or PAP. B cell function was examined by measurements of hemagglutination titers to SRBC sensitization in BALB/c females treated in two experiments similar to the ERF. T cell functions examined were highly suppressed (p less than 0.01) by PAP given hours, not days, prior to the measurement. The B cell function examined was not affected by PAP.

Effect of dietary and injectable vitamin E and selenium in weanling swine antigenically challenged with sheep red blood cells.

Two experiments were conducted to evaluate the effect of supplemental vitamin E and (or) Se, provided either in the diet or by injection, on humoral antibody production in weanling swine after an antigenic challenge with sheep red blood cells (SRBC). In the first experiment, a 2 x 2 factorial design was used, with pigs fed either 0 or .5 ppm Se and 0 or 220 IU vitamin E/kg diet. The basal diet contained a natural Se and alpha-tocopherol content of .02 ppm and 7 mg/kg, respectively. In a second 2 x 2 factorial trial, 0 or 6 mg Se and 0 or 220 mg alpha-tocopherol were injected intramuscularly into weaning pigs fed the basal diet without supplemental Se or vitamin E. A fifth treatment group was fed a positive control diet containing both nutrients (.5 ppm Se and 220 IU vitamin E/kg). In both experiments, intraperitoneal injections of SRBS (1 x 10(8)) were administered weekly, with hemagglutination titers determined at these intervals. Hemagglutination assays indicated that vitamin E and Se independently enhanced the immune response, particularly during the latter weeks of the experiment. The combination of both nutrients, provided either in the diet or via injection, resulted in a further increase in hemagglutination titers, suggesting an additive response. Pigs receiving either Se or vitamin E had higher serum concentrations of the nutrient provided. Dietary sources of these nutrients resulted in greater serum and tissue levels than did injections.

Effect of in vitro selenium supplementation on bovine sperm motility.

The effects of selenium (Se) on motility of bovine spermatozoa were evaluated in two experiments. In Experiment 1, increasing doses of Se alone or with vitamin E were found to increase motility in Se concentrations up to 1 mug/ml. However, Se alone had a greater effect than did Se with vitamin E. The sperm motility response to Se concentrations from background to 1 mug/ml on motility best fit a quadratic equation with a maximum at about .65 mug Se/ml. In Experiment 2, the effect of Se addition before and after freezing was evaluated. This experiment demonstrated that treatment of semen with Se before freezing significantly increased the post-thaw motility as compared with portions of the same ejaculate which were not treated. Treatment after thawing produced motilities which were intermediate and not significantly different from either pre-freezing supplementation or no supplementation at all. These data indicate that some semen exhibits a greater percentage of motile sperm when Se is added prior to freezing, however, no information is yet available on the fertility of Se-treated semen.

Effects of selenium supplementation on bull sperm metabolism in vitro.

An in vitro study was conducted to examine the effects of direct supplementation of diluted semen with sodium selenite on the metabolism of bovine sperm. Selenium (Se) supplementation increased the percent motility yet did not affect the percent viability of the sperm. An increase in the oxygen consumed by the sperm was associated with the increase in sperm motility. Both the adenosine triphosphate (ATP) and total adenyl nucleotide (TN) concentrations were lowered by supplementing the sperm with Se. Although changes occurred in the adenyl nucleotide pool of the Se-supplemented sperm, these changes were not reflected in the energy charge. There was no difference in the energy charge between the Se-supplemented and unsupplemented sperm. The metabolic changes caused by Se were in vitro and occurred in a short interval of time, suggesting a catalytic effect as opposed to an enzymatic effect.

Secretion of riboflavin by the porcine uterus.

A study of 10 mature crossbred, ovariectomized gilts was conducted to determine whether riboflavin secretion by porcine uteri is regulated by estrogen and progesterone. A second experiment was performed to determine whether secretion of riboflavin occurs in pregnant animals in a temporal and quantitative pattern similar to that in nonpregnant animals. In the hormonal induction experiment, it was found that animals receiving daily injections of estrogen for 5 days followed by a daily injection of a combination of estrogen and progesterone were able to secrete riboflavin in quantities comparable to those secreted by intact animals. Additionally, it was found that the secretion of riboflavin appeared to be related to onsetn of treatment with progesterone in that riboflavin secretion increased in a time-related pattern after the beginning of progesterone treatment. In a second experiment, pregnant animals secreted riboflavin in their uterine secretions in quantities and in a time pattern similar to those found in nonpregnant animals. Nonpregnant gilts in the 8th day of an estrous cycle were used for comparison to the pregnant animals in this experiment, and the quantity of riboflavin uterine secretions in the nonpregnant animals was similar to that found on day 8 in the pregnant animals. Taken together, these two experiments indicate that riboflavin is secreted under hormonal regulation by the uterus in both pregnant and nonpregnant pigs at a precisely controlled time in the progestational phase.

Suppression of lymphocyte reactivity in vitro by porcine allantoic and amniotic fluids.

Porcine allantoic and amniotic fluids from early, mid and late pregnancy were analyzed for protein constituents and ability to suppress phytohemagglutinin (PHA)-induced lymphocyte transformation. All fetal fluids contained alpha-fetoprotein as evaluated by polyacrylamide gel electrophoresis (PAGE), with the highest concentrations appearing in mid-pregnancy amniotic fluids. In addition, allantoic fluids from mid-pregnancy contained proteins assumed to be secreted by the uterus. Both allantoic and amniotic fluids from mid-pregnancy were suppressive to PHA-induced lymphocyte transformation (P<.01). Thus it was concluded that substances, potentially able to suppress the immune response locally, exist within porcine fetal fluids, but it was not determined whether this material is of solely fetal or maternal origin, or a combination.

Appearance of the uterine specific proteins following induction of ovulation in prepubertal gilts.

PIP: The appearance of the uterine specific proteins following induction of ovulation in prepubertal gilts is described. 12 gilts each at 3, 4, and 5 months of age were allotted randomly to 1 of 2 treatment groups prior to induction of ovulation: 1) saline treated and 2) human chorionic gonadotropin (HCG) treated (400 IU daily from Days 12 through 16 of the induced cycle). Ovulation was induced with HCG following treatment with pregnant mare serum gonadotropin and the day of ovulation was designated as Day 1. All the gilts were laparotomized and uteri infused with phosphate-buffered saline on Day 16 to obtain uterine protein secretions. Plasma progesterone levels on Day 11 and observations made at laparotomy indicated that only 1 gilt 3 months of age failed to ovulate. The number of corpora lutea, plasma progesterone, total recoverable uterine protein, and uterine specific protein on Day 16 were markedly affected by the age of the gilt. These same characteristics, except uterine specific protein, were additionally affected by HCG treatment. Total recoverable uterine protein and uterine specific protein in saline and HCG-treated gilts at 3, 4, and 5 months of age were 6.3 and 1.5, 10.4 and 2.8; 38.8 and 15.2, 51.6 and 15.9; 20.4 and 7.7, 47.8 and 14.6 mg, respectively. Polyacrylamide slab gel electrophoresis showed that HCG-treated gilts at 3 months of age and both saline- and HCG-treated gilts at 4 and 5 months of age generally produced the purple basic protein and the complete profile of the low molecular weight acidic proteins during the induced cycle.^ieng

Selenium/vitamin E: role in fertilization of bovine ova.

The effect of combined supplementation with selenium and Vitamin E upon fertilization of ova was evaluated in beef cattle maintained on either an adequate or inadequate nutrition. Fertilization of ova was 100% in those females receiving supplemental selenium/vitamin E and on an adequate plane of nutrition. Interaction between plane of nutrition and selenium/vitamin E was significant for percent fertility.