the use of casa (computed assisted semen analysis) as a method for environmental and toxicological...
TRANSCRIPT
The use of CASA (computed assisted semen analysis)
as a method for environmental and toxicological risk assessment
Peter MASSÁNYI, Norbert LUKÁČ, Robert STAWARZ and Ján DANKO
INTRODUCTION
DECREASE OF SEMEN QUALITY
Auger, J. et al. Decline in semen quality among fertile men in Paris during the past 20 years. New England J. Med. 1995
Carlsen, E. et al. Evidence for decreasing quality of semen during part 50 years. BMJ 1992
Jensen, TK et al. 2002: Poor semen quality may contribute to recent decline in fertility rates. Human Reproduction 17(6): 1437-1440.
SEMEN ANALYSIS• semen analysis – evaluates certain characteristics of a male's
semen and the spermatozoa contained therein• to evaluate male fertility
– whether for those seeking pregnancy or – verifying the success of vasectomy
• depending on the measurement method– just a few characteristics may be evaluated (such as with a home kit) or– many characteristics may be evaluated (generally by a diagnostic laboratory)
– collection techniques and precise measurement method influence results
• reasons for laboratory semen analysis– part of a couple's infertility investigation– after a vasectomy to verify that the procedure was successful– for testing human donors for spermatozoa donation– for animals semen analysis commonly used in stud farming and farm animal
breeding
PARAMETERS
• Spermatozoa count
• spermatozoa count (concentration) to avoid confusion with total sperm count, measures the concentration of spermatozoa in a ejaculate, distinguished from total spermatozoa count, which is the spermatozoa count multiplied with volume
• over 15 million spermatozoa per mL is considered normal (WHO, 2010)– a lower sperm count is considered oligozoospermia– a vasectomy is considered successful if the sample is azoospermia
• the average spermatozoa count today is between 20 and 40 million per mL in the Western world, having decreased by 1 – 2% per year from a substantially higher number decades ago
PARAMETERS
• Motility vs. morphology• The World Health Organization has a value of 50% and this must be measured
within 60 minutes of collection• WHO also has a parameter of vitality, with a lower reference limit of 60% live
spermatozoa– a man can have a total number of spermatozoa far over the limit of 20 million spermatozoa per
mL, but still have bad quality because too few of them are motile– however, if the spermatozoa count is very high, then a low motility (for example, less than
60%) might not matter, because the fraction might still be more than 8 million per mL– a man can have a spermatozoa count far less than 20 million spermatozoa per mL and still have
good motility, if more than 60% of those observed spermatozoa show good forward movement• A more specified measure is motility grade, where the motility of spermatozoa are
divided into four different grades:– A: spermatozoa with progressive motility – the strongest and swim fast in a straight line– B: (non-linear motility) – these also move forward but tend to travel in a curved or crooked
motion– C: non-progressive motility – they do not move forward despite the fact that they move their
tails– D: immotile and fail to move at all
Correlation analysis of heavy metals
Bull semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 0.287 0.274 0.070 0.561 0.517
Fe 1.000 0.723 -0.298 0.194 -0.060
Zn 1.000 -0.140 0.191 -0.119
Cd 1.000 -0.467 -0.117
Pb 1.000 0.419
Ni 1.000
Ram semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 0.071 0.124 0.306 0.253 -0.709
Fe 1.000 0.005 0.034 0.058 0.443
Zn 1.000 -0.016 -0.060 0.203
Cd 1.000 0.976 -0.240
Pb 1.000 -0.197
Ni 1.000
Correlation analysis of heavy metals
Boar semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 0.113 -0.533 -0.255 -0.333 -0.352
Fe 1.000 0.232 0.783 0.791 0.744
Zn 1.000 0.306 0.478 0.161
Cd 1.000 0.973 0.586
Pb 1.000 0.614
Ni 1.000
Stallion semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 -0.192 -0.274 0.070 0.561 0.517
Fe 1.000 0.723 -0.298 0.194 -0.060
Zn 1.000 -0.140 0.191 -0.119
Cd 1.000 -0.467 -0.117
Pb 1.000 0.419
Ni 1.000
Correlation analysis of heavy metals
Fox semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 0.326 0.109 -0.200 -0.854 0.534
Fe 1.000 -0.505 0.473 -0.013 0.304
Zn 1.000 -0.504 -0.238 -0.087
Cd 1.000 0.530 0.625
Pb 1.000 -0.173
Ni 1.000
Human semen
Cu Fe Zn Cd Pb Ni
Cu 1.000 0.070 0.230 -0.090 -0.470 0.140
Fe 1.000 -0.160 0.050 0.160 0.360
Zn 1.000 0.150 -0.060 -0.070
Cd 1.000 -0.040 0.020
Pb 1.000 0.100
Ni 1.000
Correlations of heavy metals to spermatozoa quality
• significant (p<0.05) correlations (r>0.67):– metals vs. pathologal spermatozoa:
• BULLS → Ni – separated flagellum • RAMS → Ni – separated flagellum; Cu – separated flagellum; Fe –
flagellum torso• STALLIONS → Cu – flagellum torso; Zn – total number of pathological
spermatozoa; Zn – flagellum torso; Fe – total number of pathological spermatozoa; Fe – flagellum ball; Cd – separated flagellum; Pb – total number of pathological spermatozoa; Pb – separated flagellum
• BOARS → medium• FOXES → Cu – other forms of pathological spermatozoa; Zn – broken
flagellum; Fe – retention of cytoplasmic drop; Cd – separated flagellum; Cd – retention of cytoplasmic drop
• RABBITS → Cu – acrosomal changes• MEN → medium: Pb – flagellum ball; Fe – other forms of pathological
spermatozoa; Cd – large head
Related publications• Slivkova, J, Popelkova, M, Massanyi, P, Toporcerova, S, Stawarz, R, Formicki, G, Lukac, N, Putala, A, Guzik, M:
Concentration of trace elements in human semen and relation to spermatozoa quality. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2009, 44, 4, 370-375.
• Massanyi, P, Weis, J, Lukac, N, Trandzik, J, Bystricka, J: Cadmium, zinc, copper, sodium and potassium concentrations in rooster and turkey semen and their correlation. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2008, 43, 5, 563-565.
• Zemanova, J, Lukac, N, Massanyi, P, Trandzik, J, Burocziova, M, Nad, P, Capcarova, M, Stawarz, R, Skalicka, M, Toman, R, Korenekova, B, Jakabova, D: Nickel seminal concentration in various animals and correlation to spermatozoa quality. JOURNAL OF VETERINARY MEDICINE A, 2007, 54, 6, 281-286.
• Massanyi, P, Trandzik, J, Nad, P, Skalicka, M, Korenekova, B, Lukac, N, Fabis, M, Toman, R: Seminal concentration of trace elements in fox and relationships to spermatozoa quality. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2005, 40, 5, 1097-1105.
• Massanyi, P, Trandzik, J, Nad, P, Lukac, N, Skalicka, M, Korenekova, B, Cigankova, V, Toman, R, Halo, M, Strapak, P: Semen concentration of trace elements in stallions and relation to the spermatozoa quality. TRACE ELEMENTS AND ELECTROLYTES, 2004, 21, 4, 229-231.
• Massanyi, P, Toman, R, Trandzik, J, Nad, P, Skalicka, M, Korenekova, B: Concentration of copper, zinc, iron, cadmium, lead and nickel in bull, ram, boar, stallion and fox semen. TRACE ELEMENTS AND ELECTROLYTES, 2004, 21, 1, 45-49.
• Massanyi, P, Trandzik, J, Nad, P, Korenekova, B, Skalicka, M, Toman, R, Lukac, N, Halo, M, Strapak, P: Concentration of copper, iron, zinc, cadmium, lead, and nickel in bull and ram semen and relation to the occurrence of pathological spermatozoa. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2004, 39, 11-12, 3005-3014.
• Massanyi, P, Trandzik, J, Nad, P, Toman, R, Skalicka, M, Korenekova, B: Seminal concentrations of trace elements in various animals and their correlations. ASIAN JOURNAL OF ANDROLOGY, 2003, 5, 2, 101-104.
• Massanyi, P, Trandzik, J, Nad, P, Korenekova, B, Skalicka, M, Toman, R, Lukac, N, Strapak, P, Halo, M, Turcan, J: Concentration of copper, iron, zinc, cadmium, lead, and nickel in boar semen and relation to the spermatozoa quality. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2003, 38, 11, 2643-2651.
OVERVIEW
• Motility • Analyzes multiple fields, up to 200 spermatozoa/field @ 20X based on microscope
configuration 10 to 80 μ chamber depth • Evaluates multiple types of motility • Sperm Vision discriminates cells from debris • World Health Organization (WHO) parameters are available for User–defined
evaluation criteria • Differentiates motility from cell drift • Reports analysis by individual cell, per field, and sample (average of all fields) • Completes analysis in less than 2 seconds per field
MATERIAL AND METHODS
Path velocity
PARAMETERS
MATERIAL AND METHODS• CASA analysis
– Makler Counting Chamber (depth 10 µm, Sefi–Medical Instruments, Germany) – parameters evaluated –
• percentage of motile spermatozoa (motility > 5 µm/s)• percentage of progressive motile spermatozoa (motility > 20 µm/s)• DCL (distance curved line; µm)• DAP (distance average path, µm)• DSL (distance straight line, µm)• VCL (velocity curved line, µm/s)• VAP (velocity average path, µm/s)• VSL (velocity straight line, µm/s)• LIN (linearity – VSL:VCL)• STR (straightness – VSL:VAP)• WOB (wobble – VAP:VCL)• ALH (amplitude of lateral head displacement, µm)• SCF (beat cross frequency, Hz)
• statistics– PC program Excel and SAS using t–test and Scheffe’s test
CASA – RESULTS: Cd
motility
progressive motility VCL
RELATED PUBLICATIONS• Slanina, T, Petrovicova, L, Miskeje, M, Knizat, L, Mirda, J, Lukac, N, Trandzik, J, Petrovicova, I, Massanyi, P: The effect of
diluent, temperature and age on turkey spermatozoa motility in vitro. JOURNAL OF APPLIED ANIMAL RESEARCH, 2014, 42, ID JAAR-2013-0244.R2, in press.
• Lukac, N, Lukacova, J, Pinto, B, Knazicka, Z, Tvrda, E, Massanyi, P: The effect of nonylphenol on the motility and viability of bovine spermatozoa in vitro. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2013, 48, 8, 973-979.
• Tvrda, E, Lukac, N, Schneidgenova, M, Lukacova, J, Szabo, Cs, Goc, Z, Gren, A, Massanyi, P: Impact of seminal chemical elements on the oxidative balance in bovine seminal plasma and spermatozoa. JOURNAL OF VETERINARY MEDICINE (HINDAWI), 2013, Article ID 125096, 8 pages. (http://www.hindawi.com/journals/jvm/2013/125096/)
• Lukac, N, Bardos, L, Stawarz, R, Roychoudhury, S, Makarevich, AV, Chrenek, P, Danko, J, Massanyi, P: In vitro effect of nickel on bovine spermatozoa motility and Annexin V-labelled membrane changes. JOURNAL OF APPLIED TOXICOLOGY, 2011, 31, 2, 144-149.
• Lukac, N, Massanyi, P, Roychoudhury, S, Capcarova, M, Tvrda, E, Knazicka, Z, Kolesarova, A, Danko, J: In vitro effect of radiofrequency electromagnetic waves on bovine spermatozoa motility. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2011, 46, 12, 1417-1423.
• Roychoudhury, S, Massanyi, P, Bulla, J, Choudhury, MD, Straka, L, Lukac, N, Formicki, G, Dankova, M, Bardos, L: In vitro copper on rabbit spermatozoa motility, morphology and mebrane integrity. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2010, 45, 12, 1482-1491.
• Roychoudhury, S, Massanyi, P, Bulla, J, Choudhury, MD, Lukac, N, Filipejova, T, Trandzik, J, Toman, R, Almasiova, V: Cadmium toxicity at low concentrations on rabbit spermatozoa motility, morphology and mebrane integrity in vitro. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2010, 45, 11, 1374-1383.
• Slivkova, J, Massanyi, P, Pizzi, F, Trandzik, J, Roychoudhury, S, Lukac, N, Dankova, M, Almasiova, V: In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2010, 45, 6, 767-774.
• Roychoudhury, S, Massanyi, P, Slamecka, J, Chlebec, I, Trandzik, J, Bulla, J, Okab, AB, Taha, TA, Salem, MH, Ayoub, MA: In vitro gossypol induced spermatozoa motility alterations in rabbits. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART B - PESTICIDES, FOOD CONTAMINANTS, AND AGRICULTURAL WASTES, 2009, 44, 7, 730-741.
• Roychoudhury, S, Jedlicka, J, Parkany, V, Rafay, J, Ondruska, L, Massanyi, P, Bulla, J: Influence of a 50 Hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rate in rabbits. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING, 2009, 44, 10, 1041-1047.
