Spermatoanalysis HELENA NEJEZCHLEBOVÁ BI6140CEN EMBRYOLOGIY PRACTICAL COURSE 1 Reproduction § the basic property of living organisms § the ability to form the basis of a system, which is the same as the founding system § allows to preserve the species and time continuity of life, to develop, increase the number of individuals, ensure the survival of the genetic lineages § sexual and asexual, or their alternation (metagenesis) § the level of reproduction is an indicator of the well-being of an organism in a given environment § an indicator of the balance of conditions in the external and internal environment of the organism § BI6140C EMBRYOLOGIE-CVIČENÍ 2 Male fertility Fertility: § basic biological property § a prerequisite for the time existence of the species § complex feature based on the ability to have healthy offsprings in the optimal number (in a given time period) § determined by a number of genes (polygenic inheritance) § heritability, (h2) of fertility indicators is low, fertility of animals is decided mainly by environmental conditions (eg. breeding conditions) § § SPG (spermiogram) = basic examination of a semen sample, evaluation of male fertility: https://www.youtube.com/watch?v=T9_XkaXCXqc § 3 Spermatozoa § head, neck, tail § the head: flattened, the nucleus with condensed chromatin; cca 2/3 of the nucleus covered by the acrosome (enzymes important tor fertilization). § the neck: short (cca 1 µm) § the tail = middle piece + principal piece + end piece; § the middle piece: axonema (arrangement of microtubules), a sheath of mitochondria. § the principal piece: axonema § 4 Sperm morphology. Schematics of human sperm. The different cell regions... | Download Scientific Diagram Spermatozoa in different animal species Sus (μm) Homo (μm) Heald length 8,7 4,0-5,0 width 4,6 2,5-3,5 Flagellum middle piece 10 5,0-6,5 main piece 30 38,5-40 Total length 48,2 47,5-51,5 Zobrazit zdrojový obrázek BI6140C EMBRYOLOGIE-CVIČENÍ Animal Science, www.ansci.wisc.edu.edu 5 Morfology abnormalities BI6140C EMBRYOLOGIE-CVIČENÍ 6 BI6140C EMBRYOLOGIE-CVIČENÍ 7 BI6140C EMBRYOLOGIE-CVIČENÍ 8 BI6140C EMBRYOLOGIE-CVIČENÍ 9 https://www.researchgate.net/figure/Morphologically-abnormal-spermatozoa-monitored_fig1_242709808 Boar (domestic pig) – morfology abnormalities BI6140C EMBRYOLOGIE-CVIČENÍ 10 Basic values of native semen in men WHO laboratory manual for the examination and processing of human semen 2010: [USEMAP] BI6140C EMBRYOLOGIE-CVIČENÍ 11 total sperm count (mil./ejaculate) 39 mil. liquefaction do 60 minut pH of liquefied sample 7,2-7,8 sperm concentration 15 mil./ml vitality (eosin staining) 58 % total motility 40 % (progressive + non progressive) progressive motility (linear) 32 % normal morphology (head, neck and tail morphology) 4 % presence of white blood cells, prokaryotic cells Note: reference values in 2009 for men: 20 mil./ml, motility 50 % (25 % progressive motility), morphology 30 %; reference values in 1960: 80 mil./ml Basic values of native semen in boars pH 7,2 sample volume > 100 ml sperm concentration > 150 mil./ml total motility > 70 % abnormal sperm morphology inc. cytoplasmatic droplets < 25 % a)normál morfology; b) abnormal acrosome; c) proximal droplet; d) distal droplet; e+f) bent flagellum https://www.researchgate.net/figure/Boar-spermatozoa-a-Normal-morphology-b-reacted-acrosome-c-proxi mal-droplet_fig2_275366299 12 Basic findings and abnormalities: 13 BI6140C EMBRYOLOGIE-CVIČENÍ § Bürker chamber = a counting-chamber device originally designed and usually used for counting blood cells. Sample evaluation using Bürker chamber (BCh): http://www.ansci.wisc.edu/jjp1/equine/lab_supplement/hemacytometer/hemocytometer%20a.gif Komůrky počítací, Bürker https://cz.vwr.com/store/product/2991822/komurky-pocitaci-burker, http://www.ansci.wisc.edu/jjp1/equine/lab_supplement/hemacytometer/Hemocytometer%20use.html 14 Sample evaluation using Bürker chamber a)evaluate the sample under a microscope as you received it. Is a dilution of the sample necessary (compare your sample with this video: https://www.youtube.com/watch?v=T9_XkaXCXqc)? Find a suitable dilution if necessary. b) b)sample dilution: mix 50 μl of the sample with ...... μl of buffered physiological saline solution in a test tube. Mix the sample well and apply under the cover glass of the Bch. Wait 3 minutes. Then evaluate the sample under the light microscope using the magnification from 200 x to 400 x. Evaluate the sample in 10 squares of Bch, unless otherwise indicated. Count the cells inside the square and on 2 sides (see graphic materials). Calculate the concentration of the sample → c) BI6140C EMBRYOLOGIE-CVIČENÍ http://www.ansci.wisc.edu/jjp1/equine/lab_supplement/hemacytometer/hemocytometer%20a.gif http://www.ansci.wisc.edu/jjp1/equine/lab_supplement/hemacytometer/Hemocytometer%20use.html 15 0,0625 mm2 http://atraktivnibiologie.upol.cz/docs/img/databaze/genetika/slides/Burkerova%20po%c4%8d%c3%adtac%c 3%ad%20kom%c5%afrka-po%c4%8d%c3%adtac%c3%ad%20s%c3%ad%c5%a5%20(Fellnerov%c3%a1%20I.).jpg BI6140C EMBRYOLOGIE-CVIČENÍ §we count sperms with heads completely inside a specified sector/syuare, or when they touch two adjacent sides (marked in bold) of the relevant square (ie only those white marked sperms, we do not count black marked sperm) §chamber depth: 0,100 mm Sample evaluation using Bürker chamber 16 Sample evaluation using Bürker chamber b) concentration assessment (c): count motile and immotile cells separately (= motile cells in 10 squares + immotile cells in 10 squares) , then calculate c: c = (total number of counted cells x dilution) / (number of squares x square area x chamber depth) c) motility assessment: i) total motility in %: total motility in % = c of motile sperm cells / total c ii) % of cell with progressive motility: evaluate min. 100 cells (optimally 200 cells), calculate % of cells with progressive movement (https://www.youtube.com/watch?v=T9_XkaXCXqc, Progressive motility = sperm swimming in a mostly straight line or in large circles = generally healthy, functioning sperm). d) morphology evaluation: evaluate min. 100 cells (optimally 200 cells), calculate % of cells with wi normal morpsholgy, calculate abnormalities of head, neck and tail separately e) record the presence of bacteria, white blood cells and ather abnormalities BI6140C EMBRYOLOGIE-CVIČENÍ 17 Informační zdroje § V. Kos a kolektiv: Příručka pro praktická cvičení z andrologie. Brno: VFU, 2019. 40 s. § Z. Vacek: Embryologie. 2006. 255 s. § Z. Věžník : Repetitorium spermatologie a andrologie, metodiky spermatoanalýzy. Brno : Výzkumný ústav veterinárního lékařství, 2004. 1 sv. (různé stránkování). § URL 1: http://humrep.oxfordjournals.org/content/16/12/2710/F1.expansion § URL 2: https://www.researchgate.net/figure/Sperm-morphology-Schematics-of-human-sperm-The-different-cell-r egions-are-indicated_fig1_260527307 § Světová zdravotnická organizace: WHO laboratory manual for the examination and processing of human semen, 2010. § další literatura u autorky BI6140C EMBRYOLOGIE-CVIČENÍ 18