1. Peter MASSÁNYI, Norbert LUKÁČ, Robert STAWARZ and Ján DANKO
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

2. INTRODUCTION

3. DECREASE OF SEMEN QUALITY
Carlsen, E. et al. Evidence for decreasing quality of semen during part 50 years. BMJ 1992
Auger, J. et al. Decline in semen quality among fertile men in Paris during the past 20 years. New England J. Med. 1995

5. Jensen, TK et al. 2002: Poor semen quality may contribute to recent decline in fertility rates. Human Reproduction 17(6):

6. 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

7. RELATION TO FERTILITY
the characteristics measured by semen analysis are only some of the factors in semen quality
30% of men with a normal semen analysis actually have abnormal spermatozoa function
conversely, men with poor semen analysis results may go on to father children
NICE (National Institute for Health and Care Excellence) guidelines – mild male factor infertility is defined as when 2 or more semen analyses have 1 or more variables below the 5th percentile, and confers a chance of pregnancy occurring naturally through vaginal intercourse within 2 years

8. 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

9. 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

10. Correlation analysis of heavy metals
Bull semen Cu Fe Zn Cd Pb Ni
1.000
0.287
0.274
0.070
0.561
0.517
0.723
-0.298
0.194
-0.060
-0.140
0.191
-0.119
-0.467
-0.117
0.419
Ram semen Cu Fe Zn Cd Pb Ni
1.000
0.071
0.124
0.306
0.253
-0.709
0.005
0.034
0.058
0.443
-0.016
-0.060
0.203
0.976
-0.240
-0.197

11. Correlation analysis of heavy metals
Boar semen Cu Fe Zn Cd Pb Ni
1.000
0.113
-0.533
-0.255
-0.333
-0.352
0.232
0.783
0.791
0.744
0.306
0.478
0.161
0.973
0.586
0.614
Stallion semen Cu Fe Zn Cd Pb Ni
1.000
-0.192
-0.274
0.070
0.561
0.517
0.723
-0.298
0.194
-0.060
-0.140
0.191
-0.119
-0.467
-0.117
0.419

12. Correlation analysis of heavy metals
Fox semen Cu Fe Zn Cd Pb Ni
1.000
0.326
0.109
-0.200
-0.854
0.534
-0.505
0.473
-0.013
0.304
-0.504
-0.238
-0.087
0.530
0.625
-0.173
Human semen Cu Fe Zn Cd Pb Ni
1.000
0.070
0.230
-0.090
-0.470
0.140
-0.160
0.050
0.160
0.360
0.150
-0.060
-0.070
-0.040
0.020
0.100

13. 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

14. 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,
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,
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,
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,
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,
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,
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,
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,
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,

15. OVERVIEW
Motility
Analyzes multiple fields, up to X 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

16. MATERIAL AND METHODS
Path velocity

18. PARAMETERS

19. MATERIAL AND METHODS CASA analysis statistics
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

20. CASA – RESULTS: Cd
motility
progressive motility
VCL

21. 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 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,
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. (
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,
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,
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,
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,
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,
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,
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,

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