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Electron microscopy of cryopreserved boar spermatozoa

with special reference to cryo-scanning electron microscopy and immunocytochemistry

Ekwall, Hans (2007). Electron microscopy of cryopreserved boar spermatozoa. Diss. (sammanfattning/summary) SLU Service/Repro, Uppsala 2007 : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae, 1652-6880 ; 2007:123
ISBN 978-91-85913-22-0
[Doctoral thesis]

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Abstract

Cryopreservation of boar spermatozoa is as yet sub-optimal, presumably owing to variation in dehydration and membrane intactness among boars. This thesis work investigated, using cryo-scanning electron microscopy (Cryo-SEM), how cooling rates below –5oC/min affect the frozen structure of boar semen in four different packages/volumes (maxi-straws and medium straws, FlatPacks [FPs] and MiniFlatPacks [MFPs]). The hypothesis tested was that the degree of dehydration differs between containers, as well as by cooling rate, which would explain differences in post-thaw viability among boars. Cryo-SEM allows the study of the fully hydrated ultrastructure of frozen sperm samples without introducing artefacts caused by fixation or processing. It further allows visualisation of the topography of the sample, including the distribution and amount/area of the frozen water which, when sublimated, leaves the partly or fully dehydrated extender and the spermatozoa intact. Furthermore, the thesis work investigated the effect of cooling/re-warming on the localisation of the hexose substrate carriers GLUT-3 and GLUT-5 and water/cryoprotectant channels (Aquaporin 7 [AQP7]) in the plasma membrane. Use of different cooling speeds and containers clearly affected the degree of (de)hydration of the extender/spermatozoa, thus explaining recorded differences in post-thaw sperm survival, but not inter-boar variation. The degree of dehydration was most favourable for the MFPs owing to their cryogenic properties. Ultrastructural analyses by SEM and transmission electron microscopy (TEM) of immunolabelling clearly showed that boar spermatozoa express the hexose/glucose transporters GLUT-3 and GLUT-5 in both the outer and the inner plasmalemma. Their distribution in the plasma membrane changed during the process of cryopreservation, particularly for GLUT-3, whose labelling decreased dramatically post-thaw. Likewise, the channel protein AQP7, involved in the transport of water and glycerol through the plasma membrane, is present in boar spermatozoa collected from the cauda epididymides and from different fractions of the ejaculate. The domain localisation changed in ejaculated spermatozoa during cryopreservation, with a clear redistribution and increasing intensity following freezing-thawing. Both hexose (GLUT-) transporters and AQP7 are markers of interest for non-empirical studies of boar semen cryopreservation.

Authors/Creators:Ekwall, Hans
Title:Electron microscopy of cryopreserved boar spermatozoa
Subtitle:with special reference to cryo-scanning electron microscopy and immunocytochemistry
Year of publishing :November 2007
Volume:2007:123
Number of Pages:57
Papers/manuscripts:
NumberReferences
ALLI. Ekwall, H. 2007: Cryo-scanning electron microscopy discloses differences in dehydration of frozen boar semen stored in large containers. Reproduction in Domestic Animals (in press) DOI No.: 10.1111/j.1439-0531.2007.00994. II. Hernandez, M., Ekwall, H., Roca, J., Vazquez, J.M., Martinez, E., & Rodriguez-Martinez, H. 2007: Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars. Cryobiology 54: 63–70. III. Ekwall, H., Hernandez, M., Saravia, F., & Rodriguez-Martinez, H. 2007: Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks. Theriogenology 67: 1463–1472. IV. Sancho, S., Casas, I., Ekwall, H., Saravia, F., Rodriguez-Martinez, H.,Rodriguez-Gil, J.E., Flores, E., Pinart, E., Briz, M., Garcia-Gil, N., Bassols, J., Pruneda, A., Bussalleu, E., Yeste, M., & Bonet, S. 2007: Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs. Reproduction 134: 111–121. V. Ekwall, H., & Rodriguez-Martinez, H. 2007: Immunocytochemical localization of aquaporin 7 (AQP7) in boar spermatozoa. (Submitted)
Place of Publication:SLU Service/Repro, Uppsala 2007
ISBN for printed version:978-91-85913-22-0
ISSN:1652-6880
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agrovoc terms:boars, semen, spermatozoa, semen preservation, cryoprotectants, freezing, cooling, artificial insemination, quality, viability, microscopy
Keywords:spermatozoa, cryopreservation, cooling rates, cryo-packages, electron microscopy (EM), Cryo-SEM, TEM, immunocytochemistry, hexose transporters (GLUT), aquaporins (AQPs), pig
URN:NBN:urn:nbn:se:slu:epsilon-1925
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-1925
ID Code:1638
Department:(VH) > Dept. of Clinical Sciences
Deposited By: Hans Ekwall
Deposited On:23 Nov 2007 00:00
Metadata Last Modified:02 Dec 2014 10:13

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