Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats

The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post-implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA).The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats.     

A simple stepwise procedure of deriving selection index with restrictions

Summary A simplified alternative procedure was derived for computing selection indices with various restrictions. The procedure involved construction of a sub-index for the unrestricted traits by the conventional methods (Smith-Hazel index approach) and construction of a sub-index for the restricted traits based on the restrospective index approach. The two subindices were then combined to obtain the desired index which met the prespecified restriction. The formulation is straightforward and easy to compute and does not involve the complicated formula as does the derivation of restricted index by the use of the Lagrange multiplier method. As a consequence of simplification, the proposed procedure is approximate in terms of optimal gain in net merit. The procedure presented can also be extended to derive an index with simultaneous imposition of multiple restrictions. Numerical examples were given to illustrate the use of this alternative approach.Animal Research Centre Contribution No. 1251     

Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli.

A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins. The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein. Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation. This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.     

Detection of human papilloma virus DNA sequences by polymerase chain reaction

We have developed a polymerase chain reaction-based procedure for reproducible detection of the E6-E7 gene in human papilloma virus DNA sequences using formalin-fixed, paraffin-embedded tissue sections. This procedure is a simple one-step procedure which does not require any elaborate hybridization following polymerase chain reaction amplification. The protocol combines modified tissue treatment and proper primer selection for efficient amplification of target DNA in a highly specific manner allowing identification in ethidium bromide-stained gels. The procedure described here is useful for a variety of tissue preparations, particularly formalin-fixed, paraffin-embedded archival tissues.     

Determination of the novocaine salt of benzylpenicillin

A simple procedure for acidometric titration of procaine benzylpenicillin after its chloroform extraction from alkaline solutions is described. Sodium benzylpenicillin does not prevent the titration.     

Microtiter assay for acetylcholinesterase

A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.     

Ultraviolet shadowing nucleic acids on nylon membranes

We describe a method for the direct visualization of nucleic acids on nylon membranes. Nylon is weakly fluorescent under short wave ultraviolet light allowing membrane-bound nucleic acids to be detected with a sensitivity of 10 ng. This procedure involves no staining or destaining of the gels prior to transfer, does not require duplicate sample lanes or blots, and does not interfere with transfer of the nucleic acid to the membrane or subsequent hybridization.     

An apparatus to retain the spatial orientation of breast biopsies

An apparatus has been designed to retain the spatial orientation of breast biopsies throughout surgery, transport and processing in the pathology laboratory. It does not interfere with regular surgical procedures and simplifies the surgeon's task of marking the orientation of a specimen. As the use of the apparatus does not involve either chemical or physical substances it is an appropriate handling procedure for those specimens requiring sensitive testing procedures such as cell culture.     

A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources

A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can be performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA.     

The effect of unequal transversion rates on the accuracy of evolutionary parsimony.

Evolutionary parsimony is an easy-to-use method of phylogenetic inference that is based on nucleic acid sequences and that does not require the assumption that evolutionary processes in the various sites on the molecule are identical. It does, however, require a parameter constraint, known as the "balanced transversion" assumption. We show that the accuracy of the procedure is fairly insensitive to moderate violations of this assumption--and that the procedure thus is applicable under more general conditions than previously thought.     

Preparation of Inositol Triphosphate from Brain: GLC of Trimethylsilyl Derivative

A simple method for the preparation of myo-inositol triphosphate from brain is described that does not require the prior isolation of phosphatidylinositol diphosphate (triphosphoinositide). A gas chromatographic procedure for detection of inositol triphosphate is reported.     

Construction and evaluation of a coronary catheter for chronic implantation in dogs

Construction of a Silastic catheter and the procedure for chronic implantation in a coronary artery in dogs is described. In addition, studies designed to evaluate whether chronic coronary artery catheterization altered coronary vascular reactivity and myocardial function are presented. The results of these studies indicate that chronic implantation of the catheter in a coronary artery of conscious dogs does not significantly interfere with the normal reactivity of the coronary vascular bed, does not compromise regional or global left ventricular function, and does not induce collateral vessel development. This technique will be useful in studies involving the neural and metabolic regulation of the coronary circulation in animals subjected to exercise and/or exercise training.     

A statistical method for correlating tRNA sequence with amino acid specificity.

A statistical method for finding the nucleotide positions in tRNA sequences that correlate with amino acid specificity has been developed. The procedure involves finding the subset of nucleotide positions and groups of positions where the marginal density of one amino acid tRNA class does not overlap that of any other amino acid class. The procedure is an application of a statistical method known as the Expectation Maximization algorithm.     

A technique to obtain fibroblast cells from skin biopsies of living bats (Chiroptera) for cytogenetic studies

We developed a procedure to obtain fibroblasts from bat skin. A small fragment of the ear is removed under ether anesthesia. This material is then cut up into small pieces and cultured in standard cell culture media. Very good quality chromosome preparations for cytogenetic studies are obtained in about three weeks. Secondary cultures can be used for other biological studies. This procedure does not require sacrificing the animals.     

SCINTILLATION SCANNING OF BRAIN TUMORS

Brain scanning by means of externally placed moving scintillation detectors has proven to be an easy accurate method for demonstrating size, shape and position of many types of brain tumors. In a series of 53, there were few false negatives and few false positives. The procedure is easy on the patient, does not require anesthesia and can be done on outpatients. It is recommended as a screening procedure for all brain tumor suspects.     

Simple and highly efficient site-specific mutagenesis, by ligation of an oligodeoxyribonucleotide into gapped heteroduplex DNA in which the template strand contains deoxyuridine

A simple and highly efficient procedure for oligodeoxynucleotide (oligo)-directed mutagenesis has been developed. In this procedure, a gapped heteroduplex DNA is first constructed and purified. The gapped heteroduplex consists of a circular 'template' strand of DNA, which contains some misincorporated deoxyuridine nucleotides, and a complementary strand which does not contain deoxyuridine, and which lacks a defined segment. Making a specific change in the sequence of the DNA within the gapped region then only requires ligation and transformation. An oligo, exactly the same length as the gap, and with the desired sequence, is synthesized, purified, and ligated directly into the gap in the heteroduplex. When this DNA is used to transform wt (ung+) Escherichia coli, about 80% of the resulting plasmids contain the sequence determined by the synthetic oligo. One gapped heteroduplex preparation can be used for many mutagenesis experiments, so that this procedure is well-suited for producing a series of defined mutations within a defined target region flanked by sites for restriction enzyme cleavage. As the method does not require a polymerase, the effects of primer displacement and polymerase infidelity are avoided.     

A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.

A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.     

La scintigraphie osseuse en trois phases pancorporelles

Performing three-phase whole-body bone scan is easy on every patient, does not increase the radiation burden to the patient and enhances the diagnostic value of the procedure. It should be performed almost routinely on every patient scheduled for a bone scan.     

Covalent coupling of sugars to liposomes

A procedure is described for the covalent coupling of p-aminophenyl-alpha-D-mannopyranoside, in the presence of carbodiimide, to a derivative of phosphatidylethanolamine (N-glutarylphosphatidylethanolamine) incorporated into the bilayers of multilamellar liposomes prepared by the dehydration-rehydration method. It appears that much of the phospholipid derivative exposed on the surface of the outer liposomal bilayer interacts with the aminosugar. The procedure is simple, does not destabilize liposomes and could be applied to other receptor-specific aminosugars.     

On the TTSC–FTSC Formulation of Standard Parsimony

Standard parsimony analysis has recently been described in a “three-taxon-like” way (the three-taxa statements for contiguous series–four-taxa statements for contiguous series, or TTSC–FTSC procedure) in order to clarify the differences between the standard approach and three-taxon analysis. It is shown that the alleged equivalence of standard parsimony analysis and the TTSC–FTSC procedure does not hold. Some minor defects of the procedure can be fixed within the TTSC–FTSC logic, but no solution is available for two basic problems: (1) the elementary three-taxon-like statements of the TTSC–FTSC procedure are highly artificial; and (2) the equivalence with standard parsimony depends on an incomplete correction for nonindependence between these statements. However, these findings do not invalidate the reported superiority of standard parsimony as a method for biological systematics.