Publications (34) View all
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Article: Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.
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ABSTRACT: In the present study, some methodological factors affecting the acrosomal staining of frozen-thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome.Animal reproduction science 11/2012; · 1.56 Impact Factor -
Article: Genetic characterization of the endangered Kiso horse using 31 microsatellite DNAs.
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ABSTRACT: In order to contribute to conservation of the endangered Kiso horse, we clarified their genetic information using 31 microsatellite DNAs, and genotyped 125 horses, 83% of the existing breed. First, we clarified the current status of the horses. The horses were confirmed to have experienced rapid loss of population causing a bottleneck, and their effective population size was much smaller than their census size. Moreover, the number of alleles (6.3), observed heterozygosity (0.674), and expected heterozygosity (0.662) were in the same range as other endangered horses all over the world. Therefore, although their inbreeding level was not so severe (F(is): -0.017), the Kiso horse is surely one of the endangered. Second, we obtained genetic information of individuals. This information allowed us to understand the genetic distance of individuals, and might help in development of a reproductive strategy concerning the genetic distance between the mating pairs. Moreover, there appeared to be 4 subpopulations of Kiso horse, and this result was in good agreement with their historical background. Third, we confirmed that the parentage test for identification using the 31 microsatellite DNAs was highly reliable (probability of exclusion: 0.999999993). This identification increases the reliability of stud certification, and is also helpful for effective management. Understanding the genetic diversity within the population and the relationships among individuals is important to ensuring effective management for maintenance of genetic variation, and this study may help in conservation of the endangered Kiso horse.Journal of Veterinary Medical Science 09/2011; 74(2):161-6. · 0.85 Impact Factor -
Article: Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls.
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ABSTRACT: The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen-stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell-permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine-phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen-stored spermatozoa were classified as having a high-grade acrosome condition before incubation, sperm tyrosine-phosphorylated proteins, including the 33-kDa tyrosine-phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41- and 33-kDa sperm tyrosine-phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine-phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen-stored sperm were classified as having a low-grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine-phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine-phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP-dependent hyperactivation for the spermatozoa of individual Japanese Black bulls.Molecular Reproduction and Development 10/2010; 77(10):910-21. · 2.53 Impact Factor -
Article: Dilution of boar ejaculates with BTS containing HEPES in place of bicarbonate immediately after ejaculation can reduce the increased inducibility of the acrosome reaction by treatment with calcium and calcium ionophore A23187, which is potentially associated with boar subfertility.
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ABSTRACT: The present study investigated whether substitution of HEPES for bicarbonate in BTS (BTS-H) used to dilute boar ejaculates immediately after ejaculation could reduce the increased inducibility of the acrosome reaction by calcium and calcium ionophore A23187. When an ejaculate was split, diluted 5-fold with regular BTS (BTS-B) and BTS-H and stored at 17 C for 12 h or 60 h, the extender or storage time had no significant influence on sperm motility or viability measured by the eosin-nigrosin method. When spermatozoa diluted serially with BTS-B and stored (36 h) were stimulated with Ca2+ (3 mM) and A23187 (0.3 microM), the proportion of spermatozoa that underwent the acrosome reaction (% acrosome reactions) significantly increased as the magnifications of dilution increased (bicarbonate content almost unchanged by dilution). By contrast, the % acrosome reactions in spermatozoa similarly diluted and stored with BTS-H decreased with the increasing magnifications of dilution (bicarbonate decreased). Sperm motility immediately after the end of incubation without A23178 tended to be lower for BTS-H than BTS-B, and the ejaculates for BTS-H had a tendency to have a lower total protein in seminal plasma than those for BTS-B. These results implied that the samples for BTS-H could be used as a model for ejaculates possibly collected during summer and showing subfertility. When an ejaculate was split, diluted serially with BTS-B and BTS-H and stored, viability measured by staining with propidium iodide was extremely similar between the 2 extenders and among the different dilution magnifications, regardless of whether spermatozoa were washed (stored for 36-66 h) or not (stored for 66-72 h). These results suggest that boar ejaculate can be stored with BTS-H at least according to the results for sperm motility and viability and that hypersensitivity of spermatozoa to Ca2+ and A23187 potentially associated with boar subfertility could be lessened by diluting ejaculates with BTS-H.Journal of Reproduction and Development 03/2010; 56(3):309-14. · 1.46 Impact Factor -
Article: Hyperactivated motility of frozen-thawed spermatozoa from fertile and subfertile Japanese black bulls induced by cyclic adenosine 3',5'-monophosphate analogue, cBiMPS.
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ABSTRACT: This study investigated whether a cyclic adenosine 3',5'-monophosphate (cAMP) analogue, cBiMPS, could induce hyperactivated motility in frozen-thawed Japanese Black bull spermatozoa and compared the ability of spermatozoa to undergo hyperactivation between fertile and subfertile bulls. Frozen-thawed spermatozoa from 3 fertile and 2 subfertile bulls were washed, suspended in BO-Hepes medium and incubated in the presence of 0.1 mM cBiMPS for up to 4 h. At 1-h intervals, the spermatozoa were examined for hyperactivated motility. The proportions of spermatozoa showing a circular swimming pattern with asymmetrical flagellar beating and those showing whiplash beating of flagella to the total number of motile spermatozoa were expressed as C% and W%, respectively. The motile spermatozoa % was barely affected by treatment with cBiMPS or the fertility status of the sperm donor, although it gradually decreased in all sperm samples during the 4-h incubation. In the fertile bulls, the C% was 0% at 0 h of incubation but rapidly increased during the 1-h incubation with cBiMPS. It then decreased slightly towards 4 h concomitantly with a gradual increase in W% towards 4 h. In the subfertile bulls, however, the cBiMPS-induced increase of C% was delayed for 1-3 h, although the incubation time-related changes in mean W% were similar between the fertile and subfertile bulls. In the vehicle controls for cBiMPS, the C% and W% were 0% throughout incubation for all the samples examined. The results suggest that hyperactivation of the flagellum can be induced by the cAMP analogue, cBiMPS, in frozen-thawed Japanese Black bull spermatozoa and that induction of hyperactivation may serve as a useful tool for detection of functional abnormality of spermatozoa from subfertile Japanese Black bulls.Journal of Reproduction and Development 10/2009; 56(1):36-40. · 1.46 Impact Factor
