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Biomedical subjects

D G Cran

Publications and source records attributed to D G Cran.

At least 19 recordsLinked to original sources
PubMed1999

Cryopreservation of flow-sorted bovine spermatozoa.

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.

Animals↗
PubMed1999

In vitro fertilization with flow-cytometrically-sorted bovine sperm.

An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available.

Animals↗
PubMed1999

Insemination of heifers with sexed sperm.

Data from inseminating 1,000 heifers consecutively with sexed sperm and 370 heifers with control sperm in 11 small field trials are summarized. Semen was from 22 bulls of unknown fertility of various beef and dairy breeds, and 6 inseminators participated. Freshly collected sperm were sexed using a MoFlo flow cytometer/cell sorter after staining sperm with the DNA-binding dye Hoechst 33342; the principle is that the bovine X chromosome has 3.8% more DNA than the Y chromosome. Accuracy approaching 90% males or females was achieved. There was little difference in pregnancy rates between sexed, unfrozen and sexed, frozen sperm. In 5 of 6 field trials, there was little difference in pregnancy rates between insemination doses of 1.0 to 1.5 x 10(6) versus 3.0 x 10(6) sexed, frozen sperm. In the most recent trials, pregnancy rates with sexed, frozen sperm were within 90% of unsexed, frozen controls that had 7 to 20 times more sperm/insemination dose; however, in a few trials, control pregnancy rates were substantially higher than with low doses of sexed sperm. There were too few inseminations per bull to test bull differences in pregnancy rates rigorously. Insemination of sexed, frozen sperm bilaterally into the uterine horns produced pregnancy rates similar to insemination into the uterine body in 4 of 5 field trials. Pregnancy rates among inseminators did not differ significantly. There was no excess embryonic death between 1 and 2 months of gestation with pregnancies from sexed sperm, and very few abortions occurred between 2 months of gestation and term. Although rigorous epidemiological studies remain to be done, calves resulting from sexed sperm appear to exhibit no more abnormalities than controls.

Animals↗
PubMed1998

Comparative motility of X and Y chromosome-bearing bovine sperm separated on the basis of DNA content by flow sorting.

A combination of flow cytometric sperm sorting of X and Y chromosome-bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33,342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 x 10(6) sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37 degrees C) and their motion video recorded for 2 min using a magnification of x 100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33,342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR.

Animals↗
PubMed1993

Confocal and fluorescence microscopic study using lectins of the distribution of cortical granules during the maturation and fertilization of pig oocytes.

A study was carried out to determine whether pig cortical granules (CGs) could be visualized using fluorescein isothiocyanate (FITC)-labelled lectins. Following labelling with FITC-labelled peanut agglutinin (FITC-PNA), fluorescent spots were observed that had a distribution during maturation and fertilization entirely consistent with that observed by electron microscopy. For the first 18 h of in vitro maturation, most of the fluorescent spots of FITC-PNA were distributed throughout the cortical cytoplasm. Thereafter, the CGs underwent centrifugal migration to form a monolayer next to the plasma membrane. Following penetration by sperm, fluorescent spots were extruded into the perivitelline space, where they aggregated forming fluorescent clumps, which subsequently formed a reticulate structure surrounding the egg. Fluorescence was gradually lost such that by 18 h after insemination none could be detected in 70% of the eggs. The results indicate that CGs in pig oocytes contain galactosyl-rich glycoconjugates and that FITC-PNA is a useful probe for their rapid visualization and examination.

Animals↗
PubMed1992

Effects of treatment with butylated hydroxytoluene on the susceptibility of boar spermatozoa to cold stress and dilution.

Boar spermatozoa acquired resistance to cold shock immediately after exposure to 2.0 mmol butylated hydroxytoluene (BHT) l-1 when Beltsville thawing solution was used as a basic diluent, as judged by motility (the proportion of motile spermatozoa) and acrosomal integrity. The concentration of BHT could be reduced to 0.2 mmol l-1 without decreasing the protective action. However, motility was altered in the presence of greater than 0.15 mmol BHT l-1. Beltsville freezing 5 (BF5) diluent was more effective than Beltsville thawing solution in protecting spermatozoa from cold shock, but addition of BHT to BF5 diluent did not affect the motility and acrosomal morphology of spermatozoa before or after cold shock. Dilution of BHT-treated spermatozoa with BF5 diluent did not restore motility and did not afford further protection against cold shock; it was detrimental to spermatozoa treated with 2 mmol BHT l-1 for greater than 15 min. Egg yolk or lecithin had a detrimental effect. When spermatozoa were treated with 0.05-0.10 mmol BHT l-1 before slow cooling to 5 degrees C, the progressive motility and acrosomal integrity were maintained better after storage for 6 days than in untreated spermatozoa.

Acrosome↗
PubMed1990

Spatial organization of repetitive DNA sequences in the bovine sperm nucleus.

During spermatogenesis, DNA in the sperm head becomes more tightly condensed as histones are replaced by protamine-like molecules. In this article, the question is asked whether, during the production of this highly differentiated cell, controls are imposed on the spatial organization of DNA within the nucleus. Heads from bull spermatozoa were isolated by a technique that removed the plasma membrane and acrosomal contents, and the DNA was induced to decondense by addition of 2-mercaptoethanol and trypsin. Under these conditions, decondensation was induced in all regions of the head. To determine whether there was any spatial restraint on packaging of the genome, three DNA probes were used (pl.709-512, containing an interspersed repetitive sequence; pCSIH, containing a copy of the major bovine centromeric statellite sequence; p18 s and p28 s, containing the 18 S and 28 S ribosomal genes) that might be expected to hybridize to different regions. Results showed that the interspersed repetitive probe hybridized to all regions of the head, whereas the ribosomal and centromeric probes hybridized to sequences that were largely confined to the equatorial region of the sperm. We conclude that organization of the genome in the bovine sperm nucleus is not random.

Animals↗
PubMed1988

Transgenes as probes for active chromosomal domains in mouse development.

Embryonic development entails a well defined temporal and spatial programme of gene expression, which may be influenced by active chromosomal domains. These chromosomal domains can be detected using transgenes which integrate randomly throughout the genome, as their expression can be affected by chromosomal position. Position effects are probably exerted most strongly on transgenes that do not contain strong promoters, enhancers or other modulating sequences. Here we have systematically explored position effects using a transgene with the weak herpes-simplex-virus thymidine-kinase promoter, linked to the readily visualized lacZ indicator gene (HSV-TK-lacZ). Each transgenic fetus with detectable expression displayed a unique lacZ staining pattern. Thus expression of this construct is apparently dictated entirely by its chromosomal position, without any construct specificity. Furthermore the transgene is faithfully transmitted to subsequent generations, allowing for systematic mapping of changes in expression during development and in adult life. These results demonstrate that transgenes can indeed be powerful tools to probe the genome for active chromosomal regions, with the potential for identifying endogenous genes involved in organogenesis and pattern formation.

Animals↗
PubMed1988

Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate.

Microinjection of inositol 1,4,5-triphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6.8 but a full cortical reaction occurring at pH8.0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTP gamma S also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.

Animals↗
PubMed1988

Effect of rapid warming of bull and rabbit semen.

Rapid warming of semen from 5 to 37 degrees C caused visible damage to the acrosomes of bull and rabbit spermatozoa. The degree and type of damage varied with the species, the bull being the more resistant. While vesiculation was observed in rabbit spermatozoa, neither warm nor cold shock resulted in this defect in the bull. Warm shock of bull spermatozoa caused acrosomal knobbing in an anterior region of the head. Spermatozoa with thread and/or droplet-like structures were frequently observed in bull semen after cold shock.

Acrosome↗
PubMed1988

Further studies on rapid dilution and warming of boar semen.

Studies have been carried out to investigate factors related to the induction of warm shock in boar spermatozoa. Rapid dilution per se caused visible damage to acrosomes when the sample contained 7.5% or more glycerol. This dilution effect was greater at lower temperatures. Acrosomal damage was greatly reduced by raising the dilution temperature from 15 to 25 degrees C, suggesting that a change in the physico-chemical characteristics of the acrosomal membrane occurred between these temperatures. During rapid dilution with warming, the dilution rate, the magnitude of the temperature change and the terminal temperature had a significant influence on acrosomal integrity; a terminal temperature of 35 degrees C was much more detrimental than one of 25 degrees C. The first sign of acrosomal damage was observed 15 sec after rapid dilution + warming and the damage was nearly maximal by 60 sec. An antioxidant, butylated hydroxytoluene (BHT), was effective against both rapid cooling and warming, while glycerol, dimethylsulphoxide and propylene glycol were ineffective in preventing warm shock.

Acrosome↗
PubMed1987

The distribution of organelles in mammalian oocytes following centrifugation prior to injection of foreign DNA.

The centrifugation of oocytes from the domestic species is a necessary prerequisite to allow visualization of nuclei for the introduction of foreign DNA. This improvement in visibility which has allowed the production of transgenic animals is accompanied by a clear stratification of the organelles. In immature oocytes from the sheep, pig, and cow, four distinct zones are formed. These comprise, lipid, membrane-bound vesicles, organelle-free cytoplasm, and mitochondria. In mature ovine oocytes, a fifth zone of smooth endoplasmic reticulum (SER) beneath that of the vesicles is formed. This SER is produced at discrete locations within the untreated cell. The potential for removal of these fractions has implications for relating patterns of protein synthesis with particular structural components. In intact oocytes, the cortical granules are located in a peripheral position beneath the plasma membrane. However, even after subjection to high centrifugal force (65,000g for 60 min), they maintain their original location. However, treatment with the cytoskeletal inhibitors, nocodozole and cytochalasin, results in rapid exocytosis after centrifugation. It is concluded that the maintenance of the spatial relationships of this organelle is mediated through the peripheral cytoskeleton.

Animals↗
PubMed1987

Distribution of spermatozoa in the mare's oviduct.

The morphology of the uterotubal junction (UTJ) and caudal isthmus during the peri-ovulatory period, and the distribution of spermatozoa within the region, were studied in 10 Pony mares. The proximal tip of the uterine horn and caudal 1-2 cm of the isthmus were removed during oestrus or shortly after ovulation from animals mated or artificially inseminated within the previous 24 h. The tissues were incised longitudinally and fixed for scanning electron microscopy. Analysis of micrographs showed deep longitudinal and oedematous folds in the preovulatory samples. After ovulation, much of the folding and oedema disappeared. There was a regional arrangement of ciliated and nonciliated cells and the cilia showed evidence of directional orientation. Occasional spermatozoa were seen in some specimens deep in furrows between folds of the UTJ.

Animals↗
PubMed1985

The distribution of actin in sheep ovaries.

The distribution of actin in sheep ovaries has been examined using immunofluorescent techniques. The germinal epithelium, which contained abundant microfibrils, fluoresced strongly while the underlying stroma was only weakly positive. During early follicular growth a band of fluorescence was associated with the base of those granulosa cells abutting onto the basal lamina. This transitional polarity of actin distribution was correlated with cell shape changes in the basal granulosa.

Actins↗