Systems for production of calves from cultured bovine embryonic cells.

The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.

Development of one-cell porcine embryos to the blastocyst stage in simple media.

Porcine embryos were flushed from mated donors and examined for cleavage stage. One- and two-cell embryos were randomly allotted to one of the five following in vitro treatments: M199 with Earle's salts, a modified Tyrode's medium (TL), TL supplemented with 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (TLH), TLH supplemented with 5.5 mM glucose (TLHG), or TLH supplemented with 5 mM glutamine (TLHGL). The bicarbonate concentration of TLH, TLHG, and TLHGL was 2 mM, compared with the 25 mM concentration in M199 and TL. Embryos in M199 and TL were incubated in 95% air:5% CO2 at 39 degrees C. Those in the remaining three treatments were incubated in air at 39 degrees C. Embryos incubated in TL and M199 did not develop past the four- to eight-cell stage, whereas the proportions of embryos developing to the compact morula or blastocyst stage by d 7 of culture in the other treatments were as follows: TLHG, 49.1%; TLHGL, 59.4%; TLH, 63.5% (P less than .005). These results indicate that porcine embryos can be cultured from the one-cell stage to blastocyst in a simple HEPES-buffered medium in air. The ability of porcine embryos to develop without supplemental CO2 may be an important finding for use in situations in which embryos must be transported for long periods before embryo transfer.

Using a cool spot to improve the thermal comfort of glassmakers.

Glassmakers monitoring a rolled plate process suffered discomfort from the high temperature environment. This was caused by the radiant heat emitted by the furnaces and associated equipment surrounding the glassmakers' working area, combined with a high air temperature. The glassmakers are responsible for monitoring the glass rolling machine to ensure that the product is manufactured to specification, and to prevent the molten glass sticking to the rollers. This study improved their thermal comfort by the construction of a cool area. They were also brought nearer to the rolling machine. This, with the addition of a cooler environment, improved their monitoring performance. The cool spot was constructed using glass and aluminium radiant heat shields, air curtains and low velocity cooling air.

Is patellectomy compatible with an Army career?

The aim of this study was to assess the impact of patellectomy on the service career of soldiers. Information on 184 individuals on whom patellectomy was performed between 1981 and 1986, was retrieved from computer records. The criterion for success of the operation was the recovery to a fully fit category. Fifty three patients (29%) regained full fitness. The outcome of patellectomies performed for chondromalacia patellae and osteoarthritis appears to be worse than that for patellar fractures.

Chronic pancreatitis in the British Army 1978-1989.

A retrospective study was made of all 28 soldiers with chronic pancreatitis first diagnosed between 1978 and 1989. All patients were male, alcohol was the aetiological factor in 90%, the mean age at diagnosis was 30 and the commonest mode of presentation was with recurrent painful episodes of pancreatitis. Endocrine and exocrine pancreatic insufficiency occurred in a quarter and a third of patients respectively and one third required surgical intervention.

Prevalence of immunity to hepatitis A in recruits to the British Army and Royal Air Force.

Between 1989 and 1992, 92% of a sample of 2790 Service recruits aged between 17 and 35 years (mean age 19 years 7 months) were found not to be immune to infection by hepatitis A virus. The proportion of males with immunity was consistently greater than that for females. There was a significantly increased probability of immunity if individuals originated from Northern England, the Midlands and Scotland, in particular the suburbs. Among male recruits there were significantly increased probabilities of immunity associated with travel to Southern and Eastern Europe or to the Tropics, and for females with travel to North West Europe or to Southern and Eastern Europe.

Immunisation of armed service medical personnel against hepatitis B infection.

Clinical and laboratory staff of the Army and RAF medical services at risk of acquiring infection with hepatitis B were immunised against the virus with a recombinant vaccine. Vaccine was administered in Service hospitals and medical centres located throughout the world. After a primary course of vaccine, 73% of personnel developed anti-HBs titres > or = 100 IU/L to hepatitis B surface antigen and were considered protected; 11% were non-responders (anti-HBs < 10 IU/l). A significantly higher proportion of females than males, and vaccinees under 40 years of age, produced a good response. Among those achieving a good response, antibody titres were higher in the younger age group and in females. After a fourth (booster) dose of vaccine, 87.2% of the poor responders and 37% non-responders, developed anti-HBs titres > or = 100 IU/L.

Nuclear transplantation in the pig embryo: nuclear swelling.

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 microns, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 microns, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.

Nuclear transplantation in early pig embryos.

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis.

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.

Exercise-induced airway narrowing in army recruits with a history of childhood asthma.

The aim of this study was to determine whether the addition of cold, dry, inspired air to maximum heart rate (MHR) exercise increased the number of subjects demonstrating abnormal airway narrowing, when applied to symptom-free army recruits with a history of asthma in childhood. One hundred recruits with a history of asthma in childhood underwent two exercise challenges to maximum heart rate, breathing either room air or cold dry air in randomized order. The percentage fall index (%FI) was calculated for each challenge, a positive result being > or = 15%. A total of 29 subjects had a positive % fall index, 12 subjects were positive to both challenges, 7 to the room air alone and 10 to the cold air challenge alone. In these subjects (n = 29), the mean (SD) % fall index to the cold air challenge was 22.7 (11.0)% compared with 16.3 (9.5)% for the room air challenge. We conclude that when employed as a screening test for abnormal airway narrowing, the addition of cold, dry, inspired air during exercise does not increase the number of subjects with a positive response, but the magnitude of the response is increased.

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitation of bovine spermatozoa by oviduct fluid.

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.

Outcomes assessment and minimally invasive surgery: historical perspective and future directions.

BACKGROUND: Outcomes assessment is being used increasingly to shape practice patterns in all areas of medicine. Although outcomes assessment is not a new concept, the widespread application of outcomes measurement for modifying practice is novel. Instead of focusing on results of interventions in highly controlled environments, outcomes studies usually report results as they occur in uncontrolled, real-world environments. Recently, the Society of American Gastrointestinal Endoscopic Surgeons (SAGES) has initiated a society-wide initiative to monitor outcomes in patients undergoing various laparoscopic operations. METHODS: Pertinent literature is reviewed as it relates to outcomes assessment. The historical background underpinning the modern interest in outcomes is outlined. Definitions of terms useful for understanding outcomes research are given. The impact of outcomes assessment on minimally invasive surgery, both positive and negative, are examined. The SAGES outcome initiative is introduced. CONCLUSIONS: Although outcomes studies usually do not provide information on the causes of observations made, they have gained in popularity because they provide information about patient perceptions of disease, disability, and treatment. Minimally invasive surgical procedures often are reported in terms of outcomes assessment because a controlled clinical trial was rendered impossible by early and widespread application of laparoscopic surgery. The SAGES outcomes initiative will provide the necessary tools for the participation of surgeons in the process of practice profiling.