Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA.

Mucopolysaccharidosis type IVA (MPS IVA) was described in 1929 by Luis Morquio from Uruguay and James Brailsford from England, and was later found as an autosomal recessive lysosomal storage disease. MPS IVA is caused by mutations in the gene encoding the enzyme, N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Reduced GALNS activity results in impaired catabolism of two glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS). Clinical presentations of MPS IVA reflect a spectrum of progression from a severe "classical" phenotype to a mild "attenuated" phenotype. More than 180 different mutations have been identified in the GALNS gene, which likely explains the phenotypic heterogeneity of the disorder. Accumulation of C6S and KS manifests predominantly as short stature and skeletal dysplasia (dysostosis multiplex), including atlantoaxial instability and cervical cord compression. However, abnormalities in the visual, auditory, cardiovascular, and respiratory systems can also affect individuals with MPS IVA. Diagnosis is typically based on clinical examination, skeletal radiographs, urinary GAG, and enzymatic activity of GALNS in blood cells or fibroblasts. Deficiency of GALNS activity is a common assessment for the laboratory diagnosis of MPS IVA; however, with recently increased availability, gene sequencing for MPS IVA is often used to confirm enzyme results. As multiple clinical presentations are observed, diagnosis of MPS IVA may require multi-system considerations. This review provides a history of defining MPS IVA and how the understanding of the disease manifestations has changed over time. A summary of the accumulated knowledge is presented, including information from the International Morquio Registry. The classical phenotype is contrasted with attenuated cases, which are now being recognized and diagnosed more frequently. Laboratory based diagnoses of MPS IVA are also discussed.

Growth hormone (GH) therapy markedly increases the motility of spermatozoa and the concentration of insulin-like growth factor-I in seminal vesicle fluid in the male GH-deficient dwarf rat.

There is increasing evidence for an important role of the somatotropic axis in male reproductive function. We investigated the effect of recombinant bovine GH (rbGH) treatment for 21 days on semen characteristics in post-pubertal GH-deficient dwarf (dw/dw) rats. Male dw/dw rats at an age of 75-80 days were divided into two groups (n = 10 per group) and injected twice per day with either rbGH (2 micrograms/g/day) or saline. While the concentration (96.4 +/- 51.3 x 10(6) per ml) and morphology of spermatozoa (spermatozoa with normal morphology 73.5 +/- 6.3%) in the dw/dw rat were within the normal range, the motility of spermatozoa was very low (27.5 +/- 11.7%), establishing a state of sub-fertility. The rbGH treatment markedly increased (p < 0.01) motility of spermatozoa (44.5 +/- 10.7%) but did not change the concentration (144 +/- 80.3 x 10(6) per ml) and morphology (spermatozoa with normal morphology 79.5 +/- 6.0%). The rbGH treatment also significantly increased the concentration of insulin-like growth factor-I (IGF-I) in blood plasma (control 389.1 +/- 65 ng/ml, rbGH 813.9 ng/ml, p < 0.001) and in seminal vesicle fluid (control 11.3 +/- 3.0 ng/ml, rbGH 16.1 +/- 5.4 ng/ml, p < 0.05). We conclude that rbGH therapy markedly increases motility of spermatozoa in sub-fertile male GH-deficient dw/dw rats. Thus, GH therapy may offer considerable potential for the treatment of impaired male reproductive performance.

IGF-I treatment increases motility and improves morphology of immature spermatozoa in the GH-deficient dwarf (dw/dw) rat.

It has recently been shown that short-term growth hormone (GH) treatment can increase the motility of spermatozoa in the GH-deficient dw/dw rat. To examine whether the effects of GH on motility of immature spermatozoa are mediated by an increase in plasma concentrations of IGF-I, we treated GH-deficient dw/dw rats with 2 microg/g/day of IGF-I using osmotic minipumps. Body weight (saline 227+/-5 g, IGF-I 253+/-4 g) and IGF-I concentrations in blood plasma (saline 472+/-19.9 ng/ml, IGF-I 986+/-43.6 ng/ml) and seminal vesicle fluid (saline 30.9+/-1.7 ng/ml, IGF-I 47.9+/-2.9 ng/ml) were significantly increased with IGF-I treatment (P<0.001), similar to the observed responses to GH therapy in our earlier study. While epididymal fluid IGF-I concentrations were not changed, IGF-I treatment significantly increased the number of immature motile spermatozoa (saline 14.4+/-3.5%, IGF-I 28.3+/-4.1%, P<0.05) and the number of spermatozoa with normal morphology (control 65.7+/-3.3%, IGF-I 75+/-1.9%, P<0.05). These data suggest that increasing the circulating concentrations of IGF-I in the GH-deficient rat can improve the motility and morphology of immature spermatozoa and thus mimic, at least in part, the effects of GH.

Standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis.

OBJECTIVES: To evaluate the accuracy and performance of the CellForm-Human automated sperm morphometry instrument (Motion Analysis Corp., Santa Rosa, CA) using different specimen preparation, staining, and analysis techniques. SETTING: Clinical and research andrology and in vitro fertilization laboratories. PATIENTS: Individuals who were undergoing semen evaluation and infertility work-up. RESULTS: The percentage of normal sperm detected by CellForm-Human was not different for washed specimens compared with nonwashed controls. Washing and resuspension to a standard concentration in medium significantly increased spermatozoan density and homogeneity on the slide. Comparison of digitization errors and morphometric measures of sperm stained by the Papanicolaou (PAP) method, the hematoxylin portion of the PAP method, and a new method developed by us, GZIN (pronounced Gee-ZIN), showed that GZIN produced larger measures for length, width, area, and perimeter, but not for width/length, and also produced less variability for most measures than PAP or hematoxylin. The number of digitization errors was significantly less for GZIN than for PAP. Visual inspection revealed that GZIN produced a more consistent stain over the entire sperm head than hematoxylin. The number of sperm analyzed affected the percentage of normal sperm detected. The % normal stabilized only after at least 200 sperm had been analyzed for each man. CONCLUSIONS: Technical variability arising from semen preparation and slide staining methods can be reduced when specimens are washed and resuspended to a standard concentration before smearing. At least 200 sperm should be analyzed to obtain a stable estimate of the percentage of normal sperm. More sperm should be analyzed if the % normal is < 50%. Morphometric measurements are more accurate and precise when sperm are stained with GZIN than when stained with PAP or hematoxylin.

A standardized test for visual analysis of human sperm morphology.

OBJECTIVES: To develop a method to train and test simultaneously a large number of observers in the practice of visual sperm morphology analysis. DESIGN: Photographs of fixed and stained sperm were prepared. Fields of suitable sperm images were selected and individual images were numbered on each negative. Two tests, which contained a total of 100 sperm images, were created. Thirty images in each test consisted of three repeats of 10 images, while 70 images in each test were unique. The tests were administered to individuals participating in an American Fertility Society postgraduate course. Sperm images were projected on a screen and participants classified each sperm using the method that was used in their own laboratory. SETTING: Postgraduate course of The American Fertility Society. RESULTS: The majority of individuals participating in the tests used some version of the World Health Organization method. The group using the Strict method reported a lower value for the percentage of normal sperm than the groups using the other methods. The variability for the percentage of normal sperm was highest for the Strict method. The degree of classification reversal, i.e., classifying a sperm as normal during one repeat and then reversing the classification during another repeat, was high for all groups (26% to 44% of the classifications). Some degree of improvement was seen from test 1 to test 2. CONCLUSIONS: It is possible to develop efficient and inexpensive methods to train observers to perform the sperm morphology assay. Such methods also enable the objective measurement of the acquisition of proficiency, comparison between different classification methods, and identification of specific differences between observers. Such methods will become more important with implementation of the Clinical Laboratory Improvement Act.

Accuracy and precision of the CellForm-Human automated sperm morphometry instrument.

OBJECTIVE: To evaluate the accuracy and precision of the CellForm-Human (CFH) automated sperm morphometry instrument (Motion Analysis Corp., Santa Rosa, CA). SETTING: Clinical and research andrology and in vitro fertilization laboratories. PATIENTS: Individuals undergoing semen evaluation and infertility work-up. RESULTS: Coefficients of variation for repeated measures of the same sperm were 1%. Coefficients of variation of normal sperm measurements were 7.4% to 12.8%, depending on the measure. Of the objects recognized as sperm by the instrument, 6.8% were debris; hence, the sperm recognition algorithms need improvement. Mean values for all CFH measures of normal sperm from specimens clinically classified as having predominantly normal, tapered, or amorphous sperm were not different; hence, the morphometry of normal sperm from normal specimens was similar to normal sperm from specimens with two different abnormalities. The instrument classified sperm as abnormal if their length or width fell outside a critical range of values recommended by the World Health Organization. Using this method, manual and CFH classification agreed unambiguously 60% of the time. When disagreement occurred, length or width marginally exceeded the range by no more than 0.1 microns. In these cases, the technician classified sperm as normal 25% of the time and classified them as abnormal 6% of the time. Because this disagreement between methods is well below the resolution of manual methods, the overall accuracy of CFH was 91% for cell type classification. CONCLUSION: At its present stage of development, the CFH instrument exceeds the accuracy and precision of most manual approaches. With improvements in sperm recognition and type classification algorithms, it could significantly improve the reliability of morphology assays in clinical and research laboratories.

Consistency of sperm morphology classification methods.

The World Health Organization (WHO) recently suggested new morphometric dimensions for normal sperm heads and new sperm head classification rules. These specifications differ from previous WHO methods and from the Kruger method for strict morphology assessment. We analyzed the WHO and Kruger sperm classification rules to determine their appropriateness as predictive linear models. We reviewed the theoretical requirements for allometric modeling, a more traditional approach for specifying the size relationships between phenotypic characters, and developed valid allometric models based on the statistical properties of over 3,700 measurements of individual sperm head dimensions. We implemented the WHO, Kruger, and Allometric methods on the same set of digitized sperm heads to study the empirical consequences of different metric requirements and classification methods. Results show that the WHO and Kruger methods are internally consistent, but specify nonconstant variance. Hence, they are inappropriate linear models. The allometric models were internally consistent and specified constant variance. Hence, they are appropriate linear models. Significant differences were found in the percentage of normal sperm between the methods. On average, the Kruger method produced the lowest value and the Allometric methods produced significantly higher values than the WHO methods, but no average difference was found between WHO methods or between Allometric methods. Nevertheless, the percentage of normal sperm did change significantly for individual specimens. These results indicate that small differences in metric requirements and classification rules can dramatically change the percentage of normal sperm. Until more rigorous definitions of sperm features can be developed, and valid statistical models can be used to describe the population characteristics of fertile sperm, the prognostic value of sperm morphology is limited.

Automated sperm morphometry analysis (ASMA) in the rabbit.

New methods of specimen preparation were developed and a new method of objective, automated sperm morphometry analysis (ASMA) was performed to reduce the technical variation in the rabbit sperm morphology assay. The optimal staining procedure was a modified GZIN stain, which allowed the ASMA instrument to accurately recognize the distal end of the sperm head and to achieve the highest sperm recognition rate (94%). Washing and resuspending sperm to a standard concentration increased the number of sperm per microscopic field in low-concentration samples and reduced the field-to-field variation in all samples. Washing also decreased the number of sperm recognition errors by the ASMA instrument. Mean metric measurements for all sperm were: length, 7.38 microns; width, 3.91 microns; width/length, 0.53; area, 22.10 microns; and perimeter, 19.20 microns. Within-specimen coefficients of variation (CVs) ranged from 0.8% to 5.5% and between-animal CVs ranged from 2.7% to 7.5%. The use of standard specimen preparation techniques and ASMA technology can significantly reduce the technical variation in the rabbit sperm morphology assay.

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.

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

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.

Automated analysis of toxicant-induced changes in rat sperm head morphometry.

An automated sperm morphometry analysis (ASMA) instrument was developed to obtain measurements of toxicant-induced changes in rat sperm head morphometry. 1,3-dinitrobenzene (1,3-DNB), a testicular toxicant known to affect sperm parameters, was used. Twelve-week-old Sprague-Dawley rats were allocated to a control (C) and to two 1,3-DNB treatment groups (T1 = 15 mg/kg; T2 = 25 mg/kg). 1,3-DNB was administered as a single dose by gavage, and animals were sacrificed 22 days after exposure. Sperm were collected, and morphology smears were made by a standard method. One hundred sperm heads were digitized on each slide, and 8 metric measurements were automatically reported. All measurements tended to decrease in a dose-dependent manner with increasing doses of 1,3-DNB. All values for total width (Wa) and interior width (W(e)) were significantly different from one another. Wa/L was significantly larger in the control than in T1 or T2, and symmetry (S = Wb/Wa) was significantly smaller in the control than in T1 or T2. Multivariate cluster analysis revealed three subpopulations that were also visually distinct. Subpopulation no. 1 was normal, based on published descriptions of normal rat sperm; subpopulation no. 2 was abnormal with a flattened curvature and a normal length; subpopulation no. 3 was abnormal with a foreshortened length and a flattened curvature. T1 and T2 contained significantly more sperm from subpopulation no. 2 and no. 3 than C (T1 = 22% and T2 = 34% vs. C = 8% by cluster analysis). C had 93% normal sperm, while the treatments had 78% and 66%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm head morphometry analysis of ejaculate and dismount stallion semen samples.

The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculate dismount sample is representative of the entire ejaculate, this sample may be utilised in determining the fertility of the ejaculate. Ejaculate samples were collected from ten stallions using an artificial vagina. Post-ejaculate dismount samples of the same ejaculate were taken from the head of the penis. A thin smear of the collected and dismount samples were prepared onto microscope slides and spermatozoa were stained for 40 min in haematoxylin. At least 200 properly digitised sperm heads from each slide were analysed using computer automated sperm morphometry analysis. The mean values for length, width, width/length, area, and perimeter were recorded from each analysis of collected and dismount samples and compared by paired t-test. The coefficients of variation of each analysis was also recorded and compared between collected and dismount samples by paired t-test. No significant differences (P > 0.10) in any measurements were found between collected and dismount samples. The mean values for all stallions for collected and dismount samples were length = 5.96 microM and 6.06 microM, width = 2.95 microM and 2.98 microM, width/length = 0.49 and 0.49, area = 13.31 microM2 and 13.65 microM2 and perimeter = 15.54 microM and 15.74 microM respectively. No significant differences were detected in the coefficients of variation of sperm head measurements from collected and dismount samples. These results indicate sperm head measurements from dismount semen are representative of those of the ejaculate. Hence, sperm head measurements of dismount samples may be viably applied to studies of fertility or in case of clinical fertility assessment. This finding will further assist in the development of normal sperm head morphometry criteria in the stallion. Clinically, a slide can be prepared in the field after natural services matings and analysed accurately and objectively by ASMA.

The effects of cryopreservation on the morphometric dimensions of caprine sperm heads.

Cryopreserved semen has been utilised in the artificial insemination of livestock species for over 40 years, even though the detrimental effects of cryopreservation on sperm function and fertility are well documented. In the present study, computer-automated sperm-head morphometry was used to determine if goat sperm-head morphometry was affected by freezing and thawing. A microscope slide was prepared from single semen samples, collected by artificial vagina, from 10 sexually active Saanen bucks. The remainder of each sample was frozen in a tris-citrate-yolk extender. After thawing, semen smears were prepared on microscope slides. All slides were stained in haematoxylin and mean sperm-head measurements of length, width, width/length, area and perimeter were determined for each slide by computer aided sperm morphometry analysis. The effects of sperm freezing on sperm-head dimensions within and among all bucks were determined. No significant (P > 0.10) freezing effect was found between fresh semen and postthaw samples for length (7.00 microns vs 7.13 microns), width (3.77 microns vs 3.87 microns), width/length (0.54 micron vs 0.54 micron), area (19.67 microns2 vs 20.57 microns2) and perimeter (18.62 microns vs 18.83 microns) when analysed across all bucks. Significant differences (P < 0.05) were however found within three bucks for area, perimeter, length and width, with the percentage increase in measurements being significantly greater than in the remaining bucks. The variability of the morphometric dimensions were not affected by freezing. The results indicate that semen freezing did not affect the overall dimensions of sperm heads across the entire population of bucks sampled. However, since sperm-head dimensions from three bucks were affected, changes in sperm-head morphometry may be indicative of spermatozoa of the semen from individuals to successfully freeze. Because the overall mean sperm-head dimensions acquired from frozen/thawed semen were not different from those of fresh semen, previously reported measurements of goat sperm heads are probably reflective of fresh semen. More importantly, retrospective studies of sperm-head morphometry and fertility may now be performed utilising extensive breeding records from frozen semen.

Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats.

The objective of the current study was to determine if sperm function is compromised in rats deficient in growth hormone (dw/dw) since a deficiency in this hormone has been implicated as a cause of lowered fertility and spermatogenic cessation in some biological models. Spermatozoa were recovered from the cauda epididymides of adult dwarf and Wistar control male rats. Spermatozoa were diluted in Medium 199 and analysed for motility. Aliquots were then fixed in phosphate buffered formalin, sperm concentration determined and morphology assessed. The mean measurements for percent motile and normal morphology were compared by Student's t-test. Sperm concentrations were compared by Mann-Whitney two sample test. The mean percentage of motile spermatozoa was significantly greater in the Wistar control group than in the dwarf group (75 +/- 1.44 vs 28 +/- 3.71%, P < 0.001). The percentage of normal sperm morphology was also greater in the Wistar control group (88 +/- 1.00 vs 75 +/- 6.9%, P < 0.001). The concentration of spermatozoa in the cauda epididymis was also significantly higher in the Wistar group. These results indicate that sperm function parameters are diminished in growth-hormone-deficient dw/dw rats. The effects of growth hormone deficiency in rats appears to be associated with compromised spermatogenesis as well as impaired sperm motility. The GH-deficient rat appears to be a suitable model for the study of the effects of GH on sperm characteristics.

Activity of angiotensin-converting enzyme (ACE) in reproductive tissues of the stallion and effects of angiotensin II on sperm motility.

A testis-specific isoform of angiotensin-converting enzyme (ACE) has been identified in a number of mammalian species. The purpose of this study was to characterize the activity of ACE in equine spermatozoa, seminal plasma, and testis. Activity of ACE was determined in seminal plasma, ejaculated and epididymal spermatozoa from mature stallions as well as from pre- and postpubertal testis. The effect of addition of angiotensin II on equine sperm motility was also evaluated. The activity of ACE in detergent extracted sperm plasma membrane was approximately 13-fold higher than that detected in seminal plasma (93.7 mU/mg versus 7.0 mU/mg protein, respectively). Activity of ACE in equine testis was significantly higher in postpubertal than in prepubertal males (3.0 mU/mg versus 0.4 mU/mg protein, respectively), and ACE activity was reduced (P<0.001) in a dose-dependent fashion by the addition of captopril. The effect of angiotensin II on sperm motility was evaluated by computer-assisted semen analysis in sperm incubated with angiotensin II (0, 1, 10, 100 nM) at 38.5 degrees C. There was no significant effect of angiotensin II on the percent motile sperm; however, there was a significant main effect of angiotensin II (P<0.01) on the kinematic parameters beat cross frequency (BCF), average path velocity (VAP), and curvilinear velocity (VCL), respectively. In addition, there were significant stallionxconcentration interactions for amplitude lateral movement (ALH), BCF, linearity (LIN), straightness (STR), and VCL. This study demonstrates that ACE activity is present in sperm membrane from ejaculated and epididymal spermatozoa and in postpubertal testis. Further studies are required to determine the role of this testis-specific enzyme.

Computer-assisted sperm head morphometry analysis (ASMA) of cryopreserved ram spermatozoa.

Normal sperm morphology has been shown to be indicative of male fertility; however, subjective methods of assessing morphology are highly variable. Computer-assisted sperm morphometry analysis (ASMA) has been developed for the objective analysis of sperm head dimensions. Developing applicable protocols for sperm head morphometry analysis increases the efficiency of these systems. The objective of the current study was to develop accurate methods for employing ASMA of ram sperm heads. Staining methods, optimal sperm sample numbers microscopic magnification and sampling variation within and between technicians were assessed. Frozen semen from 10 fertile rams was thawed and prepared on slides for morphometric analysis. Staining spermatozoa with hematoxylin and rose bengal stains yielded the best results. Ram sperm head morphometry was accurately evaluated on at least 100 spermatozoa at x 40 objective magnification. Using these techniques, a sample could be analyzed in approximately 3 min. No significant differences in sperm head measurements were detected between 2 technicians. The system properly recognized and digitized ram spermatozoa 95% of the time. The morphometric measurements of sperm heads for all rams were as follows: length = 8.08 microns, width = 4.80 microns, width:length ratio = 0.59, area = 29.13 micron 2 and perimeter = 23.93 microns. The mean within analysis coefficients of variation for all individual analyses and parameters ranged from 4.8% for length to 6.0% for area. The variation between replicate analysis was 2.4% or less for both technicians. When applying proper sample preparation and analysis procedures no differences in measurements or variation were observed between the 2 system operators.

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C.

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.

Computer automated sperm head morphometry analysis (ASMA) of goat spermatozoa.

The development of computer automated sperm morphometry analysis (ASMA) allows for the objective analysis of sperm head dimensions. A number of studies have been performed to optimize the efficiency of these systems when analyzing spermatozoa from a variety of species. In this study, frozen semen from 10 fertile goat bucks was thawed and prepared on slides for morphometric analysis to evaluate technical variation and to standardize ASMA procedures for goat spermatozoa. Methods of staining, the number of spermatozoa necessary to sample and optimal microscopic magnification were assessed. Staining for 20 min in hematoxylin (HEM) was found to be optimal. The most efficient method of analyzing goat sperm morphometry was to evaluate 100 sperm cells at x20 objective magnification. Using these techniques, a sample could be analyzed in approximately 2 min. The system properly recognized and digitized spermatozoa 96% of the time with a target recognition error rate of less than 1%. The morphometric measurements of sperm heads for all 10 bucks were the following: length = 7.69microm, width = 3.80microm, width/length ratio = 0.5, area = 22.82microm and perimeter = 20.15microm. The mean coefficients of variation (CV) for all bucks ranged from 3.4% for length to 5.8% for area. Standardized sample preparation techniques and analysis were found to improve the efficiency of ASMA.

Assessment of equine sperm mitochondrial function using JC-1.

The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.