Electrophoretic fractionation of bovine acrosomal proteins and proteinase.
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Animals↗
Biomedical subjects
D L Garner
Publications and source records attributed to D L Garner.
Living articular cartilage is not smooth. The structure of mammalian and avian joint surfaces demonstrated in vivo by immersion incident light microscopy.
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Animals↗
Effect of storage at 5 degrees C on the disc electrophoretic patterns of ovine and bovine seminal proteins.
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Animals↗
Immunocytochemical localization of acrosin on both acrosomal membranes and in the acrosomal matrix of porcine spermatozoa.
Immunocytochemical techniques were employed to determine, at the ultrastructural level, the location of acrosin in porcine spermatozoa. Antisera to highly purified porcine acrosin was produced in rabbits. The (Fab')2 fragments of the immunoglobulins were purified and conjugated with horseradish peroxidase (HRP). Washed, formaldehyde-fixed spermatozoa were reacted with the labeled antiacrosin immunoglobulins, utilizing a direct staining technique. Electron microscopy revealed that the peroxidase reaction product of HRP-antiporcine acrosin was distributed evenly over the outer acrosomal membrane of spermatozoa with intact acrosomes. The labeled antibody was also distributed evenly over the inner acrosomal membrane of cells when the overlying acrosomal structures were absent. In some spermatozoa, labeling was noted throughout the acrosomal matrix. No significant labeling was observed in control specimens when spermatozoa were exposed to HRP-antiporcine acrosin immunoglobulins that had been adsorbed previously with excess purified acrosin or exposed to HRP-conjugated rabbit antiporcine immunoglobulins. This pattern of labeling is consistent with the hypothesis that acrosin may function as a zona lysin. The observation that the outer acrosomal membrane and acrosomal matrix are labeled suggests that acrosin is not exclusively located on the inner acrosomal membrane and, thus, could participate in physiologic events other than zona penetration.
Acrosin↗
Identification and distribution of Pz-peptidases A and B in human semen.
Human semen was fractionated into fluid, particle and spermatozoal constituents using Percoll density gradient centrifugation followed by additional separation steps. All of the fractions isolated possessed both Pz-peptidase A and Pz-peptidase B activity. The effects of inhibitors on the Pz-peptidase A and B activities of all seminal fractions were similar, suggesting that hydrolysis of the Pz-peptidase was attributable solely to these two enzymes. Estimates of the activities in intact spermatozoa indicated that 1.6 +/- 0.5 mU of Pz-peptidase A and 1.6 +/- 0.7 mU of Pz-peptidase B were present per billion spermatozoa. The predominant source of Pz-peptidase B activity in semen was the ultra-low density particle fraction (110,000 X g pellet from seminal plasma), which contained 86% of the recoverable Pz-peptidase B activity. Pz-peptidase A and B activities of fluid and particle fractions isolated from azoospermic ejaculates from vasectomized donors were similar to the activities of the corresponding fractions from normal semen. This suggested that much of the Pz-peptidase A and B activities of semen originated in accessory gland secretions. The effects of EDTA, Zn2+ and Cu2+ on soluble Pz-peptidase A and B activities of particle-free seminal plasma suggested that neither was involved in the liquefaction of semen.
Adult↗
Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function.
Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.
Animals↗
Sex-Sorting mammalian sperm: concept to application in animals.
Sperm sexing can be used to produce sexed offspring with 85%-95% accuracy (Amann, 1999; Johnson and Seidel, 1999; Seidel et al 1999a). On September 1, 2000, the sale of sexed bovine sperm commented in the United Kingdom. It will be interesting to see to what degree sexed sperm penetrate the semen market. This verified sexed product sets the stage for commercialization around the world in major animal producing countries. This commercialization of sexed sperm occurred nearly 20 years after technology for accurately determining the proportion of X and Y sperm in semen was first developed at Lawrence Livermore National Laboratory. It came about due to advances in both the hardware and the software componenets of computer science, biophysic, cell biology and applied reproductive physiology plus efforts of innovative scientist. Many individuals have contributed in making semen sexing in animals a commercial reality since the research team of Bart Gledhill, Dan Pinkel, Duane Garner, Susan Lake, and Larry Johnson began following up on the first flow cytometric studies on human sperm by Friedrich Otto, Wolfgang Göhde, and Marvin Meistrich. There was also major input from personnel at USDA Beltsville Agricultural Research Center as well as scientists at Cambridge University, Atlantic Breeders Cooperative, Colorado State University and XY Inc. These include Chuck Allen, Rupert Amann, David Cran, Patrick Doyle, Mike Evans, Lisa Herickhoff, Mervyn Jacobson, Kehuan Lu, Chris Polge, Wim Rens, John Schenk, George Seidel, Glenn Welch, and many others.
Animals↗
Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide.
A new membrane-permeant DNA stain, SYBR-14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours at 36 degrees C to assess changes in cell viability. As cell death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.
Animals↗
Post-thaw bovine spermatozoal quality estimated from fresh samples.
Comparisons were made among flow cytometrically quantified populations of fresh and cryopreserved bovine spermatozoa, tri-stained with fluorophores rhodamine 123 (R123), 5- (and 6-) carboxy-4',5'-dimethylfluorescein diacetate (CMFDA), and propidium iodide (PI), and analyzed by dual parameter flow cytometry. The purpose was to find parameters in fresh semen samples that were potentially predictive of frozen sample parameters so that bulls with marginal cryopreservation capacity could be identified. Fresh and cryopreserved aliquots of semen from two sets of six bulls were semen processed in either milk (bulls 1-6) or egg yolk citrate (bulls 11-16). Membrane-damaged red (PI) and intact green (CMFDA + R123) populations were evaluated as percentages of 10,000-cell samples or numbers per straw. In milk, gated central subsets of membrane-damaged sperm cells in fresh samples and moribund cells in post-thaw samples were significantly correlated for sample percentages (r = 0.90, P = 0.014) and cell numbers per straw (r = 0.94, P = 0.006). In egg yolk citrate, fresh and frozen membrane-damaged populations were correlated (percentages: r = 0.81, P = 0.048; numbers: r = 0.88, P = 0.019). Additionally, post-thaw motility estimated by a photographic method was correlated with the number of sperm cells per straw in the intact central green subset (r = 0.98, P = 0.0006). These findings suggest that partitioning red and gree populations into smaller, central subset populations reveals significant relationships between fresh and cryopreserved bull ejaculates. The proportion of membrane-damaged spermatozoa in fresh semen seems to be predictive of the proportion of post-thaw membrane-damaged or moribund spermatozoa. The data consistently showed ejaculates of bulls 1 and 12 as having the greatest cryopreservation potentials, and bulls 4 and 16 the least. Thus, flow cytometric evaluation of fresh semen may be useful for identifying young sires with relatively poor fertilizing potential.
Analysis of Variance↗
Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa.
Functional differences among fresh 24-hour extended and cryopreserved human spermatozoa were assessed using both computer-assisted semen analysis (CASA) and flow cytometry. The objective was to determine if there were interrelationships among various qualitative parameters of the fresh and treated samples when assessed by these two automated methods. Fertile donor specimens (n = 15) were split and examined for sperm motility and curvilinear velocity using CASA within 1 hour postejaculation, after 24 hours in TEST-yolk buffer at 5 degrees C and after cryopreservation in TEST-yolk-glycerol medium. Flow cytometric analyses were performed on 24-hour extended and cryopreserved (CP) samples after fluorescent staining with rhodamine 123 to quantify mitochondrial function and carboxydimethyl fluorescein diacetate and propidium iodide to assess plasma membrane integrity. The percentages of spermatozoa with functional mitochondria and intact membranes along with the proportion of dead cells were identified and quantified by flow cytometry. Quadrant analyses of these data were used to determine the relative red and green fluorescent intensities. The initial sperm motility was correlated to the motility observed for the 24-hour stored and the CP samples. The sperm velocity of both the initial and the 24-hour extended samples was correlated to the velocity of CP samples. As for the comparison of the two automated methods for assessing seminal quality, the only sperm motion parameter that was correlated with a sperm population identified by flow cytometry (quadrant 4) was the curvilinear velocity of the sperm after 24 hours storage (r = 0.69) and after cryopreservation (r = 0.74). The present findings indicate that additional research is needed to determine if prefreeze analyses of donor sperm could be useful in predicting the post-thaw integrity of CP samples and, thereby, be useful in screening potential semen donors.
Cryopreservation↗
Effect of semen dilution on bovine sperm viability as determined by dual-DNA staining and flow cytometry.
Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 x 10(6) spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 x 10(6) spermatozoa/0.5 ml French straw. One-half of the straws for each dilution series was stored 24 hours at 5 degrees C, while the other half was cryopreserved. Spermatozoa were stained with SYBR-14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red-stained spermatozoa (PI), viable green-stained spermatozoa (SYBR-14), and moribund spermatozoa that stained both red and green (doubly-stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR-14-stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR-14-stained spermatozoa in that semen when it was cryopreserved and diluted to < 10 x 10(6) spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR-14-stained spermatozoa in the 24-hour-stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa < 10 x 10(6) spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.
Analysis of Variance↗
Effects of cryopreservation on bull sperm head morphometry.
Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.
Animals↗