Storage of boar semen.
The problems, aspects and methods of liquid storage and freeze-thawing of boar semen are discussed and a review is given on examination of spermatozoa by the recent fluorescent staining methods.
Animals↗
Biomedical subjects
L A Johnson
Publications and source records attributed to L A Johnson.
Sexing mammalian sperm for production of offspring: the state-of-the-art.
Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.
Animals↗
Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo.
Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.
Aging↗
Assuring that managed care organizations provide appropriate instructions regarding use of emergency departments.
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California↗
Simplified process for soybean glycinin and beta-conglycinin fractionation.
A simplification of the pilot-plant scale modified Nagano method yielding two protein fractions, glycinin and beta-conglycinin, by pH adjustment and ultrafiltration membrane separation was developed and compared with our pilot-plant-scale modified Nagano procedure and with a soy protein isolate pilot-plant procedure as our reference process. Two protein fractions, glycinin and beta-conglycinin, were produced from our simplified process and compared to the three protein fractions, glycinin, beta-conglycinin, and an intermediate protein mixture, produced with the modified Nagano method. The pilot-plant yields of glycinin, beta-conglycinin, and intermediate mixture fractions from the modified Nagano method were 9.4, 10.3, and 4.8% [dry basis (db)], respectively. The yield of glycinin fraction of the simplified method was 9.7% (db), and it had a protein content and purity similar to those obtained with the modified Nagano method. The yield of the beta-conglycinin fraction was 19.6% (db), which was twice that of the modified Nagano process. The protein content of beta-conglycinin was 91.6% (db), and the purity was 62.6% of the protein content, which was 9% lower in purity than the modified Nagano method. Process optimization of the simplified method suggested the best operating conditions for the membrane filtration system were 20-25 psi inlet pressure and 200-250 L/min ultrafiltration recirculation speeds.
Antigens, Plant↗
2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness.
2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non-major histocompatibility complex (MHC)-restricted killing. Recently,9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non-MHC-restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon-gamma (IFN-gamma) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase-2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.
Animals↗
High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.
Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.
Animals↗
Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification.
Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.
Actin Cytoskeleton↗
A cognitive task analysis for dental hygiene.
To be an effective assessment tool, a simulation-based examination must be able to evoke and interpret observable evidence about targeted knowledge, strategies, and skills in a manner that is logical and defensible. Dental Interactive Simulations Corporation's first assessment effort is the development of a scoring algorithm for a simulation-based dental hygiene initial licensure examination. The first phase in developing a scoring system is the completion of a cognitive task analysis (CTA) of the dental hygiene domain. In the first step of the CTA, a specifications map was generated to provide a framework of the tasks and knowledge that are important to the practice of dental hygiene. Using this framework, broad classes of behaviors that would tend to distinguish along the dental hygiene expert-novice continuum were identified. Nine paper-based cases were then designed with the expectation that the solutions of expert, competent, and novice dental hygienists would differ. Interviews were conducted with thirty-one dental hygiene students/practitioners to capture solutions to the paper-based cases. Transcripts of the interviews were analyzed to identify performance features that distinguish among the interviewees on the basis of their expertise. These features were more detailed and empirically grounded than the originating broad classes and better serve to ground the design of a scoring system. The resulting performance features were collapsed into nine major categories: 1) gathering and using information, 2) formulating problems and investigating hypotheses, 3) communication and language, 4) scripting behavior, 5) ethics, 6) patient assessment, 7) treatment planning, 8) treatment, and 9) evaluation. The results of the CTA provide critical information for defining the necessary elements of a simulation-based dental hygiene examination.
Algorithms↗
Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome-bearing spermatozoa sorted by high speed flow cytometry.
The objective of this study was to examine preimplantation development and sperm aster characteristics of bovine male and female embryos produced by using spermatozoa sorted for the X or Y chromosome. In vitro matured oocytes were inseminated at 24 h of maturation with sorted X or Y chromosome-bearing spermatozoa, using either fresh or frozen-thawed semen. Samples were taken from each sperm group 12 h post insemination (hpi), fixed, and immunostained for the microtubule cytoskeleton. Confocal microscopy enabled visualization of sperm aster formation and microtubule characteristics of each zygote during early fertilization. Cultured embryos were checked for cleavage at 30, 35, 40 and 45 hpi, embryo development was examined daily until Day 8 of culture. Blastocyst cell numbers were determined at the end of the experiments. Reanalysis of the sorted sperm cells for DNA content showed purity rates of 90.1 and 92.1% for X and Y chromosome-bearing spermatozoa, respectively. Reduced fertilization and development rates were observed when sorted spermatozoa were used compared with fresh and frozen-thawed spermatozoa. Penetration rates at 12 hpi were 39.5, 44.7, 55.9 and 79.0%, while blastocyst formation rates at Day 8 were 26.7, 26.5, 31.7 and 40.7% for X and Y chromosome-bearing spermatozoa, using fresh and frozen-thawed semen groups, respectively. Sperm aster size was larger in males than females, while the size of pronuclei and subjective grade of sperm aster quality showed no differences between sexes. In this study, a greater cleavage rate and sperm aster size in male embryos indicated a dimorphic pattern of development in male and female embryos during fertilization and first cleavage.
Animals↗
In vitro production of pig embryos: comparisons of culture media and boars.
The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.
Animals↗
Sexing mammalian sperm--overview.
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Animals↗
Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency.
Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.
Animals↗
Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA.
Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by "sort reanalysis" of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen.
Animals↗
Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.
Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.
Animals↗
Low dose insemination in synchronized gilts.
Conventional insemination techniques in pigs require 2 to 3 x 10(9) sperm/dose. When using the latest high-speed sperm-sorting technology, one can still sort only about 5 to 6 million sperm of each sex per hour. The objective of the present study was to find the minimal sperm concentration at a low-insemination volume in pigs without diminishing fertilization rate and litter size using surgical deep intra-uterine insemination (IUI). Semen from 3 boars was collected and diluted with Androhep to 5 x 10(8), 1 x 10(8), 1 x 10(7), 5 x 10(6) or 1 x 10(6) sperm/0.5 ml. In trial 1, 109 prepuberal gilts were synchronized and surgically inseminated into the tip of each uterine horn 32 h or 38 h after hCG treatment or at the time of ovulation, respectively. Pregnant gilts were allowed to go to term. Pregnancy and farrowing rates did not differ significantly except at the lowest sperm concentration if inseminated 32 h or 38 h after hCG treatment (p < 0.05). No differences were found among insemination groups for the total number of piglets, number of piglets born alive, stillborn piglets, and mummified fetuses. In trial 2, 34 gilts were inseminated as described above 32 h after hCG. Additionally, 9 gilts were inseminated once nonsurgically with 1 x 10(9) sperm as controls. Gilts were slaughtered 48 h after insemination, and embryos were recovered. Embryos were cultured in NCSU 23 (120 h), evaluated morphologically and stained with fluorescent dye (Hoechst 33342) to visualize nuclei. Recovery rates varied between 71.4% and 84.4%. Fertilization rate of the lowest sperm concentration (1 x 10(6) sperm/horn) differed significantly (p < 0.05) from all other groups. Cleavage rates at specific developmental stages did not differ. After 5 days of in vitro culture, embryos developed to morulae and blastocysts. No differences were found for these stages. In conclusion, no major differences were found between insemination groups as long as the sperm dosage was at least 10 million sperm per gilt. The low volume was sufficient for successful deep intra-uterine insemination. Embryo development was comparable to the controls.
Animals↗
In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer.
The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P<.05) were obtained when FERT-B was used as a fertilization medium for unsorted (43.4+/-5.1%) and sorted sperm (43.1+/-1.1%;), whereas in FERT-A unsorted sperm gave a cleavage rate of 17.9+/-4.4% and sorted sperm gave 30.4+/-1.4%. Additionally, cleavage rates were higher (P<.05) after fertilization with sorted sperm vs unsorted sperm, independent of fertilization medium. Cytogenetic analysis of ova revealed that more oocytes with unsorted than with sorted sperm remained in Metaphase 2 arrest (P<.05). This was also independent of the fertilization medium. Monospermic fertilization rates were the same for IVF with unsorted or sorted sperm, independent of the fertilization system, except FERT-A with unsorted sperm (P<.05). Polyspermic fertilization rates were highest in FERT-B (37.6+/-6.6). A total of 57 pigs were born from nine litters. Six litters from sexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.
Animals↗
The Internet--approaching a ubiquitous tool for dental education.
The Internet holds promise of addressing some of dentists' and dental students' needs caused by the rapid expansion of knowledge. The Internet is used primarily for communication and for courses, but the quality of Internet courses is uneven. The Internet holds potential in formal dental education for supporting information and instructional databases and as an educational administrative tool. The use of the Internet for educational purposes raises issues such as locating resources on line, ensuring information quality, and incorporation of emerging computer architecture. Some projections are offered for the future: increased instructional quality, replacement of textbooks, creating "courses" out of modules of instructional material, faculty becoming coaches, increased use of trained instructional technologists, intelligent tutoring systems, and the merging of information, instruction, and practice systems. Numerous web sites are given.
Databases as Topic↗