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Biomedical subjects

V G Pursel

Publications and source records attributed to V G Pursel.

At least 55 records · Page 3Linked to original sources
PubMed1990

Expression and performance in transgenic pigs.

Recent research clearly shows that fusion genes can be microinjected into a pronucleus of an ovum and integrate into the pig genome. Animals with such fusion genes are called 'transgenic'. The percentage of injected ova that developed into transgenic pigs varied among experiments from 0.31% to 1.73%. The percentage of transgenic pigs that expressed the fusion gene ranged from 17% to 100%. Eleven different regulatory sequences have been used for fusion genes transferred into pigs. Some of these regulatory sequences directed strong gene expression, but control over level of expression was inadequate. Other regulatory sequences directed weak expression, but imparted only brief spikes of induced expression. The predominant gene coding sequences transferred were for growth-related hormones. Elevation of growth hormone (GH) in expressing transgenic pigs enhanced plasma concentrations of insulin-like growth factor-I (IGF-I), insulin, and glucose, improved feed efficiency about 15%, and markedly reduced subcutaneous fat compared to nontransgenic siblings. Growth rate was enhanced in some transgenic GH pigs but not in others, possibly due to dietary limits. The 'over-expression' of GH was detrimental to the general health of most transgenic pigs. The most prevalent problems were lethargy, lameness, and gastric ulcers. Gilts that expressed foreign GH genes were anoestrous. Boars that expressed foreign GH genes lacked libido, but their semen was fertile when used by artificial insemination. Six different fusion genes have been transmitted from transgenic founders to progeny. Most of the transgenic pigs that produced progeny transmitted the fusion gene as an autosomal dominant trait to about half of their progeny. Regulatory sequences that will permit full control of gene expression must be developed before the full potential of gene transfer in pigs can be realized.

Animals↗
PubMed1990

Integration, expression and germ-line transmission of growth-related genes in pigs.

We have produced transgenic pigs that harbour structural genes for bovine and human growth hormone (bGH and hGH) ligated to a mouse metallothionein-I (MT) promoter, human growth hormone-releasing factor (hGRF) ligated to the MT or mouse albumin (ALB) promoter, and human insulin-like growth factor-I (hIGF-I) ligated to MT promoter. From 0.31 to 1.03% of microinjected ova developed into transgenic pigs with the various fusion genes. Foreign GH was present in plasma of 61% of the MT-hGH and 89% of the MT-bGH transgenic pigs. Two of 7 pigs with MT-hGRF and all 3 ALB-hGRF transgenic pigs had high concentrations of GRF in their plasma, but plasma concentrations of porcine GH (pGH) were not higher in GRF transgenic pigs than in littermate control pigs. In contrast, plasma concentrations at birth ranged from 3 to 949 ng hGH/ml for MT-hGH transgenic pigs and 5 to 944 ng bGH/ml for MT-bGH transgenic pigs. Presence of the foreign GH depressed endogenous pGH to non-detectable levels. In MT-bGH transgenic pigs, plasma IGF-I was elevated more than 2-fold, plasma glucose was elevated about 30 mg/dl, and plasma insulin was 20-fold higher than in littermate or sibling control pigs. Two lines of pigs expressing the MT-bGH transgene gained 11.1% and 13.7% faster, and were 18% more efficient in converting feed to body weight gain than were sibling control pigs. Expression of the MT-bGH transgene caused a marked repartitioning of nutrients from subcutaneous fat into other carcass components, including muscle, skin, bone and certain organs. The persistent excess hGH or bGH in transgenic pigs was detrimental to general health; lameness, lethargy and gastric ulcers were the most prevalent problems. Gilts that expressed the hGH or bGH transgenes were anoestrous. Germ-line transmission was obtained in 4 of 5 expressing transgenic boars and 4 of 5 nonexpressing transgenic boars and gilts. From 2% to 73% of progeny inherited a transgene from founder transgenics. All transgenic progeny of MT-hGH, MT-bGH and MT-hGRF founder males expressed the transgene if their sire also expressed the gene. The concentration of bGH or hGH in plasma of transgenic progeny was similar to the concentration present in the founder transgenic.

Animals↗
PubMed1989

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.

Agriculture↗
PubMed1989

Effect of cyclic AMP, imidazole and triiodothyronine on the rate of RNA synthesis by ejaculated ram spermatozoa.

The effects of cyclic AMP (cAMP) and of triiodothyronine (T(3)) on the enhancement of sperm motility and metabolism are well documented, and the present study was undertaken to investigate the mechanisms underlying these effects in terms of their influence on sperm RNA synthesis in vitro. Washed ram sperm were diluted to 1 40 (v/v) with incubation buffer that contained 100 mug/ml penicillin-G and 400 mg% glucose, followed by incubation at 37 degrees C for a period of 4 h. Washed ram spermatozoa incubated with graded doses of cAMP (10(-8), 10(-6) and 10(-4) M) showed significant enhancements of the rate of (3)H-uridine incorporation into RNA, with maximal effect occurring at 10(-8) M. The presence of 3.75, 7.50 or 15.00 muM T(3) also stimulated the rate of RNA synthesis by the washed ram sperm, with maximal effect occurring at 7.50 muM. On the contrary, imidazole (a compound known to stimulate phosphodiesterase activity and consequently to decrease the intracellular cAMP levels in many tissues) was found to cause consistent inhibition of spermatozoal RNA synthesis. The inhibition was 47, 90 and 92% of control for 10, 50 and 100 mM imidazole, respectively. The results obtained indicate that cAMP may act either as a first or a second messenger in the mature sperm. The data also indicate that T(3) (possibly mediated by cAMP) may act on the ram sperm by the induction of enzymes, which are required for the well-known effects of this hormone in enhancing the sperm metabolic activity.

Journal Article↗
PubMed1989

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.

Animals↗
PubMed1988

Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa.

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.

Journal Article↗
PubMed1987

Absorption of compounds in medium by the oil covering microdrop cultures.

Microdrops of medium under either paraffin or silicone oil are commonly used for culture of early mammalian embryos. Radiolabeled estradiol, progesterone, and androstenedione in drops of medium under oil decreased by 51, 89, and 77%, respectively, after 24-hr incubation. Up to 14% of labeled estradiol moved to another drop of medium by passing through the oil. Several other substances tested (FSH, leucine, glucose, lactate, sodium ion, PGE2, PGF2 alpha) did not pass into the oil. Both paraffin and silicone oils can alter the composition of culture medium by absorbing and transferring certain types of compounds.

Absorption↗
PubMed1987

Duration of thawing on post thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws.

Ejaculates from six boars were frozen in 5-ml maxi-straws and stored in liquid nitrogen. Maxi-straws were thawed by submersion in a 52 degrees C circulating waterbath for 28, 34, 40, 46, 52, or 58 sec. The post thaw percentages of motile sperm and acrosomes with a normal apical ridge (NAR) at the initial microscopic examination were significantly higher (P < 0.05) for maxi-straws submerged for 40 sec than for any other duration of submersion. The mean core temperature of maxi-straws after 40 sec of submersion was 4.8 degrees C. Based on regression equations for post thaw motility and NAR acrosomes, a 37 sec thawing time would be expected to provide the maximal post thaw survival for boar sperm frozen in maxi-straws.

Journal Article↗
PubMed1987

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.

Animal Husbandry↗
PubMed1986

Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana).

Electroejaculates from free-ranging, African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity. In Study I, each ejaculate was tested with each of 7 cryoprotective diluents. After cooling to 5 degrees C and equilibration on ice (4 degrees C) for 120 min, each aliquant was pellet frozen on solid CO2, stored in liquid nitrogen and thawed (37 degrees C) in saline or tissue culture solution. Amongst all diluents, post-thaw sperm motility, motility duration in vitro (37 degrees C) and acrosomal integrity were greatest (P less than 0.05) when diluent BF5F was used. Thawing medium had no effect on results. In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling-equilibration interval in an electronic cooler (5 degrees C); however, post-thaw sperm motility rating and duration of motility in vitro were greater (P less than 0.01) with the pellet than the straw container freezing method. When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12 h by maintaining thawed semen at 21 rather than 37 degrees C (P less than 0.05). All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (approximately 1.5 degrees C/min) compared to that of Study I (approximately 6.5 degrees C/min). This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa must effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.

Acrosome↗
PubMed1986

Genetic engineering of mammalian embryos.

A gene consisting of the mouse metallothionein I promoter/regulator (MT) fused to the human growth hormone (hGH) structural gene (MThGH) was microinjected into rabbit, sheep and pig eggs. Visualization of nuclear structures was accomplished by interference-contrast (I-C) microscopy for rabbit and sheep eggs and by centrifugation and I-C microscopy for pig eggs. The gene integrated into the chromosomes of each species with an efficiency of 13% in rabbits, 1% in sheep and 10% in pigs. Human GH mRNA was detected in the liver of transgenic rabbits as well as tail and ear samples of pigs. Immunoreactive hGH was present in the serum of a transgenic rabbit and plasma of most transgenic pigs. In several pigs hGH levels increased between birth and 90 d of age. The presence of substantial quantities of hGH in plasma of pigs did not increase postnatal somatic growth rates. Founder animals will be bred and their transgenic and control progeny used to assess the effects of hGH on feed efficiency and carcass composition. These experiments demonstrate the feasibility of introducing foreign genes into the genome of several animal species by microinjection of eggs.

Animals↗
PubMed1985

Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei.

The obscured pronuclei or nuclei in living one- and two-celled pig ova were revealed after centrifugation for 3 min at 15,000 X g. To determine viability of centrifuged ova, one- and two-celled pig ova were collected from superovulated gilts; half of the ova were centrifuged and all ova were transferred into recipient gilts. Prior to transfer all embryos were stained with tetramethylrhodamine isothiocyanate (TRITC) to distinguish the experimental embryos from the recipients's own ova. Centrifuged ova were transferred into one oviduct of recipient gilts and uncentrifuged ova were deposited into the opposite oviduct. Embryos were recovered 4 days after transfer and were stained with lacmoid or Hoechst 33342 to assess their stage of development. Centrifugation had no detectable influence on survival of the recovered embryos to 4 days. Centrifugation is a simple, reliable method for revealing pronuclei and nuclei of one- and two-celled pig ova and apparently does not alter subsequent preimplantation development.

Animals↗
PubMed1984

Effect of incubation conditions, inhibitors and seminal plasma on protein synthesis in ram spermatozoa.

The effects of incubation time (15 min-4 h), rate of semen to buffer dilution (1/10-1/40), and concentration of glucose (5.5-22 mM) on the rate of protein synthesis by ejaculated washed ram spermatozoa were determined. The rate of protein synthesis increased linearly as incubation time, dilution rate, and the glucose concentration increased. Denaturation of sperm protein with 1% HgCl2 caused an almost complete inhibition of amino acid incorporation. Protein synthesis over a period of 4 h was also inhibited by chloramphenicol but was not affected by cycloheximide. Protein synthesis and uptake of [14C]cAMP by washed ram spermatozoa was also significantly inhibited by the inclusion of 2-8% seminal plasma in the buffer. The present results indicate that the authentic protein synthesis by mature ram spermatozoa is mainly of mitochondrial origin. The data also suggest a role for intracellular cAMP in the regulation of sperm protein synthetic activity.

Animals↗
PubMed1983

Effect of processing of semen on capacitation time of fresh and frozen-thawed boar spermatozoa.

Ovulation time of gilts was controlled by oral treatment with altrenogest (17-allyl-hydroxyestra-4,9,11-trien-3-one) followed by gonadotropic hormone injections. Gilts were laparotomized 42 to 44 h after injection of human chorionic gonadotropin. Spermatozoa were surgically placed into ligated oviducts of gilts after one of the following semen treatments: frozen and thawed in Beltsville thawing solution (F-BTS) or in seminal plasma (F-SP); unfrozen, processed like the frozen semen, but not cooled, frozen or thawed, and extended in Beltsville thawing solution (U-BTS) or in seminal plasma (U-SP). Ova were recovered 5 or 24 h after insemination and their stage of development was determined. At 5 h, F-BTS and U-BTS spermatozoa had fertilized significantly more ova (45 and 69%, respectively) than the F-SP and U-SP spermatozoa (5 and 34%, respectively). Ova fertilized by F-BTS and U-BTS spermatozoa had reached a more advanced stage of development than ova fertilized by F-SP and U-SP spermatozoa at both recovery times. Unfrozen spermatozoa (U-BTS or U-SP) fertilized more ova by 5 h after tubal deposition than F-BTS or F-SP spermatozoa (P less than .005 for each comparison). By 24 h, the percentage of fertilized ova was similar for all treatments. The results indicate that semen processing reduced capacitation time of boar spermatozoa to an extent similar to passage through the uterus. The capacitation time of frozen-thawed spermatozoa appeared to be similar to spermatozoa that were processed, but not cooled, frozen or thawed.

Animals↗
PubMed1982

Effect of uterine ligation and cervical plugs on retention of frozen-thawed boar sperm.

Two experiments were conducted to determine whether the prevention of retrograde sperm expulsion from the uterus would enhance ovum fertilization by frozen-thawed boar sperm and whether insertion of a cervical plug would reduce the loss of frozen-thawed boar sperm from the uterus. In Exp. 1, 25 gilts were surgically inseminated and one uterine horn was ligated to prevent expulsion of sperm. Nine gilts were killed 4 h after insemination, and the sperm were recovered from the uterine horns and uterotubal sections. Significantly more sperm were recovered from the ligated than from the unligated uterine horns. Fertilized ova were recovered from nine of 16 gilts killed 48 to 90 h after insemination. More ova (P less than .005) were fertilized on the ligated than on the unligated sides of the reproductive tracts (87 vs 64%). In Exp. 2, 24 gilts were artificially inseminated with frozen-thawed boar sperm and then randomly selected to receive the intracervical insertion of a cotton tampon, a plastic plug or no treatment (control). Sperm were recovered from the uterine horns and uterotubal sections 4 h after insemination. The cervical plugs failed to significantly increase the retention of sperm in the uterotubal sections and uterine horns.

Animals↗
PubMed1981

Comparison of various extenders for freeze-preservation of semen from selective captive wild ungulates.

Semen collected by electroejaculation from selected captive wild ungulates was used to evaluate the cryoprotective ability of 4 semen diluents. Three species of ungulates were used: blesbok (Damaliscus dorcas phillipsi), dorcas gazelle (Gazella dorcas), and onager (Equus hemionus onager). Fresh semen was immediately evaluated for the percentage of motility and the progressive motility, and an aliquot was fixed for evaluation of the acrosomal integrity of spermatozoa. The remaining volume of ejaculate was divided into equal aliquots and either frozen raw on dry ice or extended with each of the 4 diluents. Samples were concurrently equilibrated at 5 C, frozen on dry ice, and stored in liquid nitrogen. Aliquots were thawed for evaluation of motility traits and spermatozoal acrosomes. Each diluent provided cryoprotection in each species. However, spermatozoa varied in ability to survive cryopreservation, and postthaw viability was influenced by the semen diluent used. Although ineffective in the onager, the study of morphologic forms of spermatozoal acrosomes proved useful for assessing the cryoprotective property of semen diluents in the blesbok and dorcas gazelle.

Acrosome↗
PubMed1981

Synchronization of estrus in gilts with allyl trenbolone: fecundity after natural service and insemination with frozen semen.

The synthetic progestogen, allyl trenbolone, was fed daily to 60 gilts at 15 mg/gilt for 18 days. Sixty-eight gilts that came into estrus the same week as treated gilts served as controls. Gilts were checked for estrus twice daily and were artificially inseminated with frozen semen or bred by natural service. All but two treated gilts returned to estrus between 4 and 7 days after withdrawal of allyl trenbolone (mean = 5.6; SD = .82). The farrowing rate among gilts inseminated with frozen semen was significantly lower than that among gilts bred by natural service (52.5 vs 89.6%). The farrowing rate among synchronized gilts was similar to that among untreated gilts (70.7 vs 73.5%). Average total and live litter sizes at birth and litter size at weaning were significantly smaller for untreated gilts inseminated with frozen semen than for synchronized gilts inseminated with frozen semen or those of untreated gilts bred by natural service. Average litter size for synchronized gilts bred by natural service was not significantly larger than that for synchronized gilts inseminated with frozen semen or that of untreated gilts bred by natural service.

Animals↗