Biomedical subjects

W V Holt

Publications and source records attributed to W V Holt.

At least 37 records · Page 2Linked to original sources

PubMed1999

Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species.

After mating, inseminated spermatozoa are transported to the oviduct. They attach to and interact with oviductal epithelial cells (OEC). To investigate sperm-OEC interactions, we used chlortetracycline to study the capacitation status of boar spermatozoa in coculture with homologous OEC and cells of nonreproductive origin (LLC-PK1, porcine kidney epithelial cell line). Boar spermatozoa were cocultured with OEC and LLC-PK1 cells for 15, 60, 120, or 240 min. The proportion of capacitated spermatozoa in coculture with the isthmic and ampullar cells increased significantly (p < 0.05) during incubation. However, most spermatozoa in coculture with LLC-PK1 cells or blank (medium only) remained uncapacitated. In addition, preferential binding of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa to OEC and the other cell type was investigated. Our approach was to vary the proportions of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa in suspension using long preincubation and lysophosphatidylcholine treatment of semen prior to a very short incubation with OEC or LLC-PK1 cells. The results showed that the majority of spermatozoa that were bound to OEC or LLC-PK1 cells were uncapacitated and that a significant relationship existed between the relative proportion of uncapacitated spermatozoa in the control samples and those bound to LLC-PK1 cells (r2 = 0.43, p < 0.005). However, there was no correlation between the proportion of uncapacitated spermatozoa in the control samples and the proportion of those bound to isthmic or ampullar cells. In conclusion, the results clearly demonstrated the specific nature of the sperm-OEC interaction in the porcine species. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by the induction of capacitation in cocultured spermatozoa.

Animals↗

PubMed1999

Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system.

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace-Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0-22.0 microns, 22.1-73.0 microns and 73.1-130.0 microns). The Landrace-Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1-130 microns long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean +/- SD; 66.36 +/- 24.70 microns) to the Landrace-Meishan introgression boar (mean +/- SD; 67.09 +/- 21.80 microns). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.

Animals↗

PubMed1999

Role of reproductive technologies and genetic resource banks in animal conservation.

In combination with modem reproductive technologies, there is potential to use frozen and stored germplasm (genetic resource banks) to support conservation measures for the maintenance of genetic diversity in threatened species. However, turning this idea into reality is a complex process, requiring interdisciplinary collaboration and clearly defined goals. As the number of species deserving the attention of conservation scientists is overwhelmingly large, yet detailed knowledge of reproductive physiology is restricted to relatively few of them, choosing which species to conserve is one of the most difficult issues to be tackled. Besides the direct application of technologically advanced reproductive procedures, modern approaches to non-invasive endocrine monitoring play an important role in optimizing the success of natural breeding programmes. Through the analysis of urine and faecal samples, this type of technology provides invaluable management information about the reproductive status of diverse species. For example, it is possible to diagnose pregnancy and monitor oestrous cycles in elephants and rhinos without causing stress through restraint for sample collection. In this review, we identify the potential contribution of reproductive biology and genetic resource banks to animal conservation, but also highlight the complexity of issues determining the extent to which this potential can be achieved.

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↗

PubMed1998

Non-invasive diagnosis of pregnancy in wild black rhinoceros (Diceros bicornis minor) by faecal steroid analysis.

The objective of this study was to determine whether faecal progestagen measurement could be used to diagnose pregnancy in wild black rhinoceros cows. Immunoreactive 20alpha-progestagens were measured in faecal samples collected regularly (one or two times times per week) from pregnant and non-pregnant wild black rhinoceros females (n = 6) in Zimbabwe. Fresh dung piles deposited by the study animals were serially sampled during prolonged periods of tracking with local game scouts. Samples were stored frozen, and dried prior to methanol extraction. Immunoreactivity in faecal extracts was measured with a 20alpha-dihydroprogesterone enzyme immunoassay and was shown to reflect circulating progesterone concentrations. Mean concentrations of faecal 20alpha-progestagens during each month of gestation were significantly higher than faecal concentrations in non-pregnant animals (P<0.05), except during the second month of gestation. Faecal 20alpha-progestagens remained 5-10 times higher than concentrations in non-pregnant animals from the 4th to 15th month of gestation. It was concluded that regular non-invasive reproductive monitoring of black rhinoceros in the wild was possible and that pregnancy could be accurately diagnosed from the measurement of 20alpha-progestagens in faecal samples. The use of this technique in wild black rhinoceros populations will offer new perspectives for in situ management of this endangered species.

20-alpha-Dihydroprogesterone↗

PubMed1997

Alternative strategies for the long-term preservation of spermatozoa.

A number of perceived future requirements for stored germplasm in agriculture, aquaculture, biotechnology and conservation are discussed in the present review. In the light of these broad demands, it is apparent that current approaches to gamete and embryo storage need considerable improvement, and that novel approaches to the technologies of germplasm preservation should be pursued if possible. The present article is presented in response to a request for novel future research ideas in this area. Early literature on desiccation, and later research into natural mechanisms of survival during desiccation, is considered in relation to the development of freeze-drying and vitrification methods. Developments in reproductive technologies themselves may mean that freeze-dried spermatozoa could realistically be used for direct intracytoplasmic microinjection of oocytes. Other radical methods that may achieve germplasm preservation are harvesting testicular cells in culture, cryopreserving immature or seasonally regressed testicular material, or a combination of both. Evidence from non-mammalian testicular culture systems, and recent success with the transmeiotic development of mouse spermatids in vitro, suggest that these approaches may eventually become feasible.

Animal Husbandry↗

PubMed1997

Assessment of boar sperm function in relation to freezing and storage.

The functions necessary for normal fertilization to occur in vivo or in vitro are examined and a rational approach to identifying the main features of a fertilizing spermatozoon are developed. It is concluded that methods for testing the quality of spermatozoa must probe the dynamic changes experienced by the spermatozoa during capacitation or under stressful incubation conditions. Recent developments in the multivariate analysis of sperm motility data are used to illustrate the success that can be achieved by this approach. Ideally, changes in sperm motility characteristics should be correlated with an assessment of capacitation status. However, until the capacitation status of any individual cell can be clearly defined this will remain problematic.

Animals↗

PubMed1996

Angiotensin II stimulates sperm motility.

The physiological factors which induce and maintain mammalian sperm maturation and motility generally remain unclear, although several agents are known to be involved. We recently described the application of immunocytochemical and immunoblotting methods to identify the angiotensin II type 1 (AT1) receptor in the tails of ejaculated rat and human sperm, and gave evidence to show that angiotensin II may promote sperm motility. These data are extended here by the application of a computerised sperm tracking system (the Hobson Sperm Tracker) to demonstrate that AII has actions on specific motility parameters, including curvilinear velocity, straight line velocity, and amplitude of lateral head movement. Since local tissue renin-angiotensin systems are present in both male and female tracts, the data suggest that angiotensin II has a role in the maintenance of sperm function and fertility.

Angiotensin I↗

PubMed1996

Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation.

Sampling conditions that affect the biological validity of computer-assisted analysis of ram sperm motion were examined using a continuous real-time computerized semen analysis system (Hobson Sperm Tracker). Search radius (SR, 10 settings) and minimum track point (MTP, 10 settings) were varied factorially to evaluate their effects on the inclusion of sperm subpopulations within derived datasets. Low SR (< 12 microns) or high MTP values (> 26 frames) precluded measurements of rapidly moving cells, whereas high SR (> 17 microns) and low MTP settings (< 22 frames) led to erroneous tracking and poor data quality. Suitable settings for these set-up parameters were derived and tested for their biological consistency with four methods of preparing ram semen for computerized analysis. The preparation techniques tested were: centrifugation through sucrose-based Ficoll and Percoll media, a swim-up technique and simple dilution in Tyrode's media. The "selective' Percoll and swim-up methods generated sperm populations with significantly higher linear velocities and a lower tendency to deviate from linear trajectories than from either the Ficoll method or dilution technique. Deleterious effects of centrifugation were evident, particularly on sperm survival in vitro over several hours. It is concluded that computer-assisted semen analysis provides useful information about the behaviour of ram spermatozoa in vitro, but the measurement conditions must be defined carefully.

Animals↗

PubMed1996

Oestrous synchronization, semen preservation and artificial insemination in the Mohor gazelle (Gazella dama mhorr) for the establishment of a genome resource bank programme.

Gazella dama mhorr is an endangered species with an extant population of about 190 animals distributed between several zoos. Semen was collected by electro-ejaculation from 12 adult males, and cryopreserved in TEST-yolk diluent containing 6% glycerol. The effects of the concentration of egg yolk (5%, 10% and 20%) and the presence or absence of sodium triethanolamine lauryl sulfate (equex) on sperm motion and acrosomal integrity after thawing were examined. Increasing concentrations of egg yolk resulted in more acrosomal damage and poorer motility after thawing. The presence or absence of equex had no effect on either parameter. The frozen spermatozoa were used in an insemination trial, in which 13 females were treated with intravaginal progesterone-releasing devices to synchronize oestrus. Seven females were inseminated with frozen-thawed semen 48 h after removal of the devices, and six were inseminated after 60 h. Three females in the first group and one in the second group became pregnant. However, only one pregnancy (from the 48-h group) was carried to term. The study demonstrated the feasibility of applying artificial insemination in this species, but revealed that a number of outstanding technical problems remain to be solved.

Acrosome↗

PubMed1994

Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasma membrane damage in cryopreserved ram spermatozoa.

The objective of this investigation was to examine the nature of freeze/thaw-induced plasma membrane damage in an effort to validate hypotheses about cryoinjury in ram spermatozoa. Spermatozoa were loaded with fluorescein diacetate (FDA), a marker for plasma membrane integrity, and cooled (15 degrees C/min) to temperatures between -10 degrees C and -30 degrees C on a cryomicroscope stage. Post-thaw fluorescence intensity measurements of individual cells indicated that freezing to temperatures between -10 degrees C and -15 degrees C did not induce significant membrane permeabilization. However, freezing below -15 degrees C was followed by membrane permeabilization immediately after thawing. A majority (> 60%) of flagellar plasma membranes of cells frozen to -10 degrees C remained ultrastructurally intact during thawing; principal-piece membranes were more robust than middle piece membranes (p = 0.001). Significant middle-piece membrane breakage was, however, induced as the post-thaw temperature increased from +10 degrees C to +30 degrees C (10 degrees C, 64 +/- 12.3% intact membranes [mean +/- SEM]; 30 degrees C, 43 +/- 12.5% intact membranes [mean +/- SEM]; p = 0.0085). Cells frozen to -30 degrees C did not exhibit this thawing effect, although the distinction between middle-piece and principal-piece plasma membranes was evident (p = 0.002). All sperm head plasma membranes were damaged by freezing and thawing to any combination of temperatures. Although acrosomes became swollen after freezing and thawing, the incidence of outer acrosomal membrane vesiculation remained at control (unfrozen) levels with all treatments used. Experimental exposure to the hyperosmotic conditions generated during freezing induced little flagellar membrane permeabilization, but significant damage was caused by restoration of osmotic equilibrium. It is suggested that membranes are initially destabilized during the freezing process, both by low temperature effects and by exposure to high salt concentrations. The resultant post-thaw degeneration of the plasma membrane is caused by a combination of temperature and osmotic effects.

Acrosome↗

PubMed1992

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.

Animals↗

PubMed1991

Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa.

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.

Actins↗

PubMed1990

Sperm motility, velocity and migration.

Four different methods for evaluating sperm motility were analysed for experimental error: subjective assessment of a wet film preparation, sperm velocity measured by time-lapse photography, sperm velocity measured by computer analysis and sperm migration across a nucleopore membrane. Subjective assessment of motility was found to be inaccurate, within single observer and between 2 observers. Both methods of measuring mean sperm velocity were accurate, particularly that using the computer analysis system; a high technical failure rate was found using time-lapse photography. Sperm migration across a nucleopore membrane was found to be highly inaccurate. Two groups were then analysed for the predictive value of these tests (excluding sperm migration): 104 proven fertile men and 53 infertile men. Although subjective motility was able to predict from which group the sample came at optimum cut-off with 78% accuracy, computer analysed sperm velocity could predict with overall 91% accuracy at optimum cut-off. Computer analysis of sperm velocity offers a rapid, objective and predictive assessment of sperm function.

Humans↗

PubMed1989

The value of sperm swimming speed measurements in assessing the fertility of human frozen semen.

The purpose of this study was to evaluate relationships between measured sperm velocity and in-vivo fertility, using donor semen samples from an artificial insemination (AID) programme. Seventy-one frozen semen samples were examined; measurements of sperm velocity were made immediately after thawing, upon a motile 'swim-up' fraction, and finally after 3.5 h incubation at 37 degrees C in the freezing mixture. Zona-free hamster egg penetration assays were performed upon all samples. Two groups of samples were identified; seven donors (11 samples) had failed to produce any pregnancies through AID from a range of 3 to 14 cycles tested, whilst the remaining samples (from 25 donors) had achieved at least one pregnancy each. The mean sperm velocity (+/- SEM) for the latter 'fertile' group was significantly higher than the corresponding value for the 'infertile' group; (i) after thawing, 65.9 +/- 1.8 versus 50.4 +/- 3.2 microns/s (P less than 0.001) and (ii) after 3.5 h incubation, 42.1 +/- 2.1 versus 24.7 +/- 5.7 microns/s (P less than 0.002). Using the maintenance of sperm velocity during incubation as an indicator of survival, life-table analyses were used to calculate monthly conception rates on various sub-groups of the semen samples. Poor survival (greater than 40% decline in velocity over 3.5 h) was associated with a monthly pregnancy rate of only 11.58% (362 cycles), whilst better survival (less than 40% decline) was associated with the significantly higher (P = 0.024) pregnancy rate of 16.87% (480 cycles).

Evaluation Studies as Topic↗

PubMed1988

Direct observation of cold-shock effects in ram spermatozoa with the use of a programmable cryomicroscope.

Cryoinjury in individual ram spermatozoa was investigated in cells cooled at 10 degrees C/min on a programmable cryomicroscope. In physiological buffer and cryoprotective media, there was a smooth decline in sperm swimming speed with decreasing temperature; cooling in buffer caused a marked decline in the proportion of cells displaying forward progression, especially once the temperature fell below 16 degrees C. Spermatozoa cooled in the presence of rhodamine 123, a mitochondrial-specific dye, showed that abolition of sperm motility by cold shock in buffer was not due to mitochondrial inactivation. Temperature decline through the region of 10 degrees C caused a number of spermatozoa in buffer to undergo a sudden asymmetric bending of the flagellum in the region of the midpiece. Ultrastructural studies suggest that this was caused by an unstable, asymmetric membrane lesion. Spermatozoa cooled in the presence of cryoprotective media showed better recovery of motility after rewarming and failed to exhibit the bending effect described above.

Animals↗

PubMed1988

The role of membrane-active lipids in the protection of ram spermatozoa during cooling and storage.

The interactions of ram spermatozoa with exogenous liposomes of varying composition were studied, with the aim of examining the mechanisms by which some lipids protect against cold-induced damage during cryostorage. Liposomes containing various preparations of phosphatidylcholine and cholesterol enhanced sperm survival during storage at 5 degrees C, both in terms of motility and acrosomal integrity. A membrane-fluidizing agent, A2C, was slightly deleterious in this respect. Cholesterol-containing liposomes were not superior in their effects to those prepared without cholesterol. Thus stabilization of the plasma membrane by cholesterol loading may be unimportant. When sodium vanadate was used as a functional probe of membrane integrity, the cryoprotective effects of lipids were apparent despite increased plasma membrane permeability. Incubation of spermatozoa with positively charged liposomes, containing stearylamine, caused considerable loss of motility and acrosomal damage, coupled with cellular aggregation. There was also some evidence that the presence of calcium lessened the effectiveness of liposomes in protecting spermatozoa against damage during cooling.

Albumins↗

PubMed1988

Administration of a gonadotropin releasing hormone analogue in oligozoospermic infertile males.

This study investigated the stimulatory potential of a superactive gonadotropin releasing hormone analogue in idiopathic oligozoospermia. In a double-blind trial, 19 men were randomized to receive buserelin (D-Ser(TBU)6-GnRH ethylamide) in one of two dosages or saline, twice weekly, for twelve weeks. Treatment did not lead to a significant increase in serum concentrations of the pituitary gonadotropins or in sperm concentrations. However, in the dosages and schedules investigated, there was no inhibitory effect of the analogue. Further assessment of this approach is suggested.

Buserelin↗