The effect of heterologous seminal plasma from ram, buck or camel on the freezability of ram semen

https://doi.org/10.17221/52/2018-VETMEDCitation:Swelum A.A., Saadeldin I.M., Bahadi M., Afifi M., Al-Mutary M., Alowaimer A.N. (2018): The effect of heterologous seminal plasma from ram, buck or camel on the freezability of ram semen. Veterinarni Medicina, 63: 500-512.
download PDF

Here, we compared the effects of ram, buck and dromedary camel seminal plasma mixed with TRIS-egg yolk glycerol extender on the freezing preservation of ram semen. Awassi ram semen samples underwent primary evaluation and were then pooled and diluted with the following diluents: TRIS-egg yolk glycerol mixed with (1) whole ram semen as a control (T); (2) ram sperm after seminal plasma removal (W); or (3) ram, (4) buck or (5) camel seminal plasma (R, B and C, respectively). The diluted semen was frozen using liquid nitrogen vapor. Various sperm parameters were evaluated in the frozen semen. Total motility before and after freezing was significantly higher in R, B and C diluents than in T and W diluents. Progressive motility after freezing was significantly higher in R, B, C and T diluents than in W diluent. Vitality after freezing was significantly higher in B than in W diluent. DNA fragmentation before and after freezing was significantly lower in R, B, C and T diluents than in W diluent. Plasma membrane integrity before and after freezing was significantly higher in R, B and C diluents than in W diluent. Sperm abnormalities before freezing were significantly lower in R, B and C diluents than in W diluent. Malondialdehyde concentration was significantly higher in T and W diluents than other diluents. Reduced glutathione concentration was significantly higher in B diluent than other diluents. Moreover, reduced glutathione concentration was significantly higher in C, R and W diluents than in T diluent. Thus, the addition of ram, buck or camel seminal plasma to TRIS-egg yolk glycerol extender improved the quality of frozen ram semen, while seminal plasma removal adversely affected it. Ram, buck and camel seminal plasma had similar effects, with no significant differences between them on the evaluated parameters of frozen ram semen.

References:
Agarwal A. (2005): Prevention of Oxidative Stress Injury to Sperm. Journal of Andrology, 26, 654-660  https://doi.org/10.2164/jandrol.05016
 
Aitken RJ (1995): Free radicals, lipid peroxidation and sperm function. Reproduction, Fertility and Development, 7, 659-  https://doi.org/10.1071/RD9950659
 
Aitken R.John, Buckingham Donna W., Carreras Alfonso, Stewart Irvine D (1996): Superoxide dismutase in human sperm suspensions: Relationship with cellular composition, oxidative stress, and sperm function. Free Radical Biology and Medicine, 21, 495-504  https://doi.org/10.1016/0891-5849(96)00119-0
 
Aitken RJ, Wingate JK, De Iuliis GN, Koppers AJ, Mclaughlin EA (2006): Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. Journal of Clinical Endocrinology & Metabolism 91, 4154–4163.
 
Alghamdi A S, Foster D N, Troedsson M H T (2004): Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Reproduction, 127, 593-600  https://doi.org/10.1530/rep.1.00096
 
Arienti G., Carlini E., Palmerini C.A. (1997): Fusion of Human Sperm to Prostasomes at Acidic pH. Journal of Membrane Biology, 155, 89-94  https://doi.org/10.1007/s002329900160
 
Ashworth PJ, Harrison RA, Miller NG, Plummer JM, Watson PF (1994): Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reproduction, Fertility and Development, 6, 173-  https://doi.org/10.1071/RD9940173
 
Aurich J.E., Kühne A., Hoppe H., Aurich C. (1996): Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology, 46, 791-797  https://doi.org/10.1016/S0093-691X(96)00237-3
 
Barbas J. P., Mascarenhas R. D. (2009): Cryopreservation of domestic animal sperm cells. Cell and Tissue Banking, 10, 49-62  https://doi.org/10.1007/s10561-008-9081-4
 
Barrios Beatriz, Pérez-Pé Rosaura, Gallego Margarita, Tato Agustín, Osada Jesús, Muiño-Blanco Teresa, Cebrián-Pérez José A. (2000): Seminal Plasma Proteins Revert the Cold-Shock Damage on Ram Sperm Membrane1. Biology of Reproduction, 63, 1531-1537  https://doi.org/10.1095/biolreprod63.5.1531
 
Barrios B. (2005): Immunocytochemical Localization and Biochemical Characterization of Two Seminal Plasma Proteins That Protect Ram Spermatozoa Against Cold Shock. Journal of Andrology, 26, 539-549  https://doi.org/10.2164/jandrol.04172
 
Bergeron Annick, Villemure Michèle, Lazure Claude, Manjunath Puttaswamy (2005): Isolation and characterization of the major proteins of ram seminal plasma. Molecular Reproduction and Development, 71, 461-470  https://doi.org/10.1002/mrd.20310
 
Bilodeau Jean-Fran�ois, Chatterjee Suvro, Sirard Marc-Andr�, Gagnon Claude (2000): Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development, 55, 282-288  https://doi.org/10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7
 
BRADLEY MARK P., FORRESTER IAN T. (1982): Human and Ram Seminal Plasma Both Contain a Calcium-Dependent Regulator Protein. Journal of Andrology, 3, 289-296  https://doi.org/10.1002/j.1939-4640.1982.tb00686.x
 
Brouwers Jos F.H.M, Gadella Barend M (2003): In situ detection and localization of lipid peroxidation in individual bovine sperm cells. Free Radical Biology and Medicine, 35, 1382-1391  https://doi.org/10.1016/j.freeradbiomed.2003.08.010
 
Cardozo J.A., Fernández-Juan M., Forcada F., Abecia A., Muiño-Blanco T., Cebrián-Pérez J.A. (2006): Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. Theriogenology, 66, 841-850  https://doi.org/10.1016/j.theriogenology.2006.01.058
 
Chatterjee Suvro, Gagnon Claude (2001): Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Molecular Reproduction and Development, 59, 451-458  https://doi.org/10.1002/mrd.1052
 
Chaudhari D. V., Dhami A. J., Hadiya K. K., Patel J. A. (2015): Relative efficacy of egg yolk and soya milk-based extenders for cryopreservation (−196°C) of buffalo semen. Veterinary World, 8, 239-244  https://doi.org/10.14202/vetworld.2015.239-244
 
Lamirande E. De, Gagnon C. (1993): A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. International Journal of Andrology, 16, 21-25  https://doi.org/10.1111/j.1365-2605.1993.tb01148.x
 
Domínguez M.P., Falcinelli A., Hozbor F., Sánchez E., Cesari A., Alberio R.H. (2008): Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm. Theriogenology, 69, 564-573  https://doi.org/10.1016/j.theriogenology.2007.11.010
 
Einspanier Ralf, Krause Ingolf, Calvete Juan J., Töfper-Petersen Edda, Klostermeyer Henning, Karg Heinrich (1994): Bovine seminal plasma ASFP: Localization of disulfide bridges and detection of three different isoelectric forms. FEBS Letters, 344, 61-64  https://doi.org/10.1016/0014-5793(94)00362-9
 
El-Hajj Ghaoui R, Thomson PC, Evans G, Maxwell WMC (2004): Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion. Reproduction in Domestic Animals, 39, 173-180  https://doi.org/10.1111/j.1439-0531.2004.00499.x
 
El-Hajj Ghaoui R, Thomson PC, Leahy T, Evans G, Maxwell WMC (2007): Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen?Thawed Ram Spermatozoa. Reproduction in Domestic Animals, 42, 541-549  https://doi.org/10.1111/j.1439-0531.2006.00819.x
 
El-Manna M.M., Tingari M.D., Ahmed A.K. (1986): Studies on camel semen. II. Biochemical characteristics. Animal Reproduction Science, 12, 223-231  https://doi.org/10.1016/0378-4320(86)90043-6
 
Forouzanfar M., Sharafi M., Hosseini S.M., Ostadhosseini S., Hajian M., Hosseini L., Abedi P., Nili N., Rahmani H.R., Nasr-Esfahani M.H. (2010): In vitro comparison of egg yolk–based and soybean lecithin–based extenders for cryopreservation of ram semen. Theriogenology, 73, 480-487  https://doi.org/10.1016/j.theriogenology.2009.10.005
 
FUKUI Yutaka, KOHNO Hirohide, TOGARI Tetsuro, HIWASA Mami, OKABE Kentaro (2008): Fertility after Artificial Insemination Using a Soybean-Based Semen Extender in Sheep. Journal of Reproduction and Development, 54, 286-289  https://doi.org/10.1262/jrd.20004
 
García-López N., Ollero M., Muiño-Blanco T., Cebrián-Pérez J.A. (1996): A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma. Theriogenology, 46, 141-151  https://doi.org/10.1016/0093-691X(96)00149-5
 
Garner D.L., Thomas C.A., Gravance C.G., Marshall C.E., DeJarnette J.M., Allen C.H. (2001): Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology, 56, 31-40  https://doi.org/10.1016/S0093-691X(01)00540-4
 
Gil J (2003): Fertility of ram semen frozen in Bioexcell® and used for cervical artificial insemination. Theriogenology, 59, 1157-1170  https://doi.org/10.1016/S0093-691X(02)01178-0
 
Gunay U, Dogan I, Nur Z, Manolov I, Sagirkaya H, Soylu MK, Kaptan C, Akpinar L (2006): Influence of bull seminal plasma on post-thaw ram semen parameters and fertility. Bulletin-Veterinary Institute in Pulawy 50, 503–507.
 
Guthrie H. D., Welch G. R. (2007): Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid1. Journal of Animal Science, 85, 1402-1411  https://doi.org/10.2527/jas.2006-787
 
Hafez E, Hafez E (1993): Semen evaluation. Reproduction in Farm Animals 5, 405–423.
 
Iwamoto Teruaki, Tanaka Hiroki, Osada Takao, Shinagawa Toshihito, Osamura Yoshiyuki, Gagnon Claude (1993): Origin of a sperm motility inhibitor from boar seminal plasma. Molecular Reproduction and Development, 36, 475-481  https://doi.org/10.1002/mrd.1080360411
 
Jobim M, Bustamante FI, Trein C, Wald V, Gregory R, Mattos R (2005): Equine seminal plasma proteins related with fertility. Animal Reproduction Science 89, 305–308.
 
Juyena N. S., Stelletta C. (2012): Seminal Plasma: An Essential Attribute to Spermatozoa. Journal of Andrology, 33, 536-551  https://doi.org/10.2164/jandrol.110.012583
 
Kawano N, Shimada M, Terada T (2004): Motility and penetration competence of frozen–thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing. Theriogenology, 61, 351-364  https://doi.org/10.1016/S0093-691X(03)00218-8
 
Kelso K. A., Cerolini S., Speake B. K., Cavalchini L. G., Noble R. C. (1997): Effects of dietary supplementation with  -linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerel from 24 to 72 weeks of age. Reproduction, 110, 53-59  https://doi.org/10.1530/jrf.0.1100053
 
Killian Gary J., Chapman David A., Rogowski Lee A. (1993): Fertility-Associated Proteins in Holstein Bull Seminal Plasma1. Biology of Reproduction, 49, 1202-1207  https://doi.org/10.1095/biolreprod49.6.1202
 
Marti E., Marti J. I., Muino-Blanco T., Cebrian-Perez J. A. (2008): Effect of the Cryopreservation Process on the Activity and Immunolocalization of Antioxidant Enzymes in Ram Spermatozoa. Journal of Andrology, 29, 459-467  https://doi.org/10.2164/jandrol.107.003459
 
Martins Jorge André Matias, Souza Carlos Eduardo Azevedo, Silva Fredy David Albuquerque, Cadavid Verónica Gonzalez, Nogueira Fabio César, Domont Gilberto Barbosa, de Oliveira José Tadeu Abreu, Moura Arlindo A. (2013): Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Research, 113, 115-127  https://doi.org/10.1016/j.smallrumres.2013.01.005
 
Maxwell W.M.C., Watson P.F. (1996): Recent progress in the preservation of ram semen. Animal Reproduction Science, 42, 55-65  https://doi.org/10.1016/0378-4320(96)01544-8
 
Maxwell WMC, de Graaf SP, Ghaoui R El-Hajj, Evans G (2007): Seminal plasma effects on sperm handling and female fertility. Reproduction in Domestic Ruminants, 6, 13-38  https://doi.org/10.5661/RDR-VI-13
 
Melo Luciana Magalhães, Teixeira Dárcio Ítalo Alves, Havt Alexandre, da Cunha Rodrigo Maranguape Silva, Martins Danyelly Bruneska Gondim, Castelletti Carlos Henrique Madeiros, de Souza Paulo Roberto Eleutério, Filho José Luiz de Lima, Freitas Vicente José de Figueirêdo, Cavada Benildo Sousa, Rádis-Baptista Gandhi (2008): Buck (Capra hircus) genes encode new members of the spermadhesin family. Molecular Reproduction and Development, 75, 8-16  https://doi.org/10.1002/mrd.20757
 
Ménard Martin, Nauc Veronica, Lazure Claude, Vaillancourt Denis, Manjunath Puttaswamy (2003): Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid. Molecular Reproduction and Development, 66, 349-357  https://doi.org/10.1002/mrd.10369
 
Mortimer S. T., Maxwell W. M. C. (1999): Kinematic definition of ram sperm hyperactivation. Reproduction, Fertility and Development, 11, 25-  https://doi.org/10.1071/RD99019
 
Moura Arlindo A., Chapman David A., Koc Hasan, Killian Gary J. (2007): A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls. Animal Reproduction Science, 98, 169-188  https://doi.org/10.1016/j.anireprosci.2006.03.012
 
Moura Arlindo A., Souza Carlos E., Stanley Bruce A., Chapman David A., Killian Gary J. (2010): Proteomics of cauda epididymal fluid from mature Holstein bulls. Journal of Proteomics, 73, 2006-2020  https://doi.org/10.1016/j.jprot.2010.06.005
 
Neild D.M., Brouwers J.F.H.M., Colenbrander B., Agüero A., Gadella B.M. (2005): Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa. Molecular Reproduction and Development, 72, 230-238  https://doi.org/10.1002/mrd.20322
 
Ollero M, Cebrian-Perez JA, Mui O-Blanco T (1997): Improvement of cryopreserved ram sperm heterogeneity and viability by addition of seminal plasma. Journal of Andrology 18, 732–739.
 
Park NC, Park HJ, Lee KM, Shin DG (2003): Free radical scavenger effect of rebamipide in sperm processing and cryopreservation. Asian Journal of Andrology 5, 195–201.
 
Pérez-Pé R., Cebrián-Pérez J.A., Muiño-Blanco T. (2001): Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa. Theriogenology, 56, 425-434  https://doi.org/10.1016/S0093-691X(01)00574-X
 
Pérez-Pé Rosaura, Grasa Patricia, Fernández-Juan Marta, Peleato Maria Luisa, Cebrián-Pérez José Álvaro, Muiño-Blanco Teresa (2002): Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa. Molecular Reproduction and Development, 61, 226-233  https://doi.org/10.1002/mrd.1152
 
Peris Soliman I., Bilodeau Jean-François, Dufour Maurice, Bailey Janice L. (2007): Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Molecular Reproduction and Development, 74, 878-892  https://doi.org/10.1002/mrd.20686
 
Ratto MH, Huanca W, Singh J, Adams GP (2005): Local versus systemic effect of ovulation-inducing factor in the seminal plasma of alpacas. Reproductive Biology and Endocrinology 3, doi: 10.1186/1477-7827-3-29. https://doi.org/10.1186/1477-7827-3-29
 
Rebolledo AD (2007): Fertility in hair sheep inseminated with freeze spermatozoa rediluited with seminal plasma. Revista Cientifica 17, 73–76.
 
Robertson Sarah A. (2005): Seminal plasma and male factor signalling in the female reproductive tract. Cell and Tissue Research, 322, 43-52  https://doi.org/10.1007/s00441-005-1127-3
 
Rodrigues M.A.M., Souza C.E.A., Martins J.A.M., Rego J.P.A., Oliveira J.T.A., Domont G., Nogueira F.C.S., Moura A.A. (2013): Seminal plasma proteins and their relationship with sperm motility in Santa Ines rams. Small Ruminant Research, 109, 94-100  https://doi.org/10.1016/j.smallrumres.2012.07.032
 
Salamon S., Maxwell W.M.C. (1995): Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination. Animal Reproduction Science, 37, 185-249  https://doi.org/10.1016/0378-4320(94)01327-I
 
Salamon S, Maxwell W.M.C (2000): Storage of ram semen. Animal Reproduction Science, 62, 77-111  https://doi.org/10.1016/S0378-4320(00)00155-X
 
Kei Satoh (1978): Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clinica Chimica Acta, 90, 37-43  https://doi.org/10.1016/0009-8981(78)90081-5
 
Schöneck C., Braun J., Einspanier R. (1996): Sperm viability is influenced in vitro by the bovine seminal protein aSFP: Effects on motility, mitochondrial activity and lipid peroxidation. Theriogenology, 45, 633-642  https://doi.org/10.1016/0093-691X(95)00409-2
 
Setchell B, Brooks D (1988): Anatomy, vasculature, innervation and fluids of the male reproductive tract. In: Knobil E, Neill J (eds): The Physiology of Reproduction. Raven Press, New York. 753–836.
 
Sharma L, Pandey V, Nigam R, Saxena A, Swain DK, Yadav B (2016): Association of oxidative status and semen characteristics with seminal plasma proteins of buffalo semen. Iranian Journal of Veterinary Research 17, 226–230.
 
Simpson A. M., Swan M. A., White I. G. (1987): Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine. Gamete Research, 17, 355-373  https://doi.org/10.1002/mrd.1120170408
 
Smith Ivan K., Vierheller Thomas L., Thorne Carol A. (1988): Assay of glutathione reductase in crude tissue homogenates using 5,5′-dithiobis(2-nitrobenzoic acid). Analytical Biochemistry, 175, 408-413  https://doi.org/10.1016/0003-2697(88)90564-7
 
Souza Carlos Eduardo A., Moura Arlindo A., Monaco Elisa, Killian Gary J. (2008): Binding patterns of bovine seminal plasma proteins A1/A2, 30kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid. Animal Reproduction Science, 105, 72-89  https://doi.org/10.1016/j.anireprosci.2007.11.027
 
Swelum AA, Saadeldin I, Alanazi MB, Afifi M, Ba-Awadh H, Alowaimer AN (2018a): Effects of adding egg yolks of different avian species to Tris glycerol extender on the post-thawing quality of buck semen. Animal Reproduction Science 195, 345–354.
 
Swelum AA, Saadeldin I, Ba-Awadh H, Alowaimer AN (2018b): Effects of melatonin implants on the reproductive performance and endocrine function of camel (Camelus dromedarius) bulls during the non-breeding and subsequent breeding seasons. Theriogenology 119, 18–27.
 
Taha T. A., Abdel-Gawad E. I., Ayoub M. A. (2000): Monthly variations in some reproductive parameters of Barki and Awassi rams throughout 1 year under subtropical conditions 1. Semen characteristics and hormonal levels. Animal Science, 71, 317-324  https://doi.org/10.1017/S1357729800055168
 
Teixeira D.I., Cavada B., Sampaio A., Havt A., Bloch Jr. C., Prates M., Moreno F.B., Santos E., Gadelha C.A., Gadelha T., Crisostomo F.S., Freitas V. (2002): Isolation and Partial Characterisation of a Protein from Buck Seminal Plasma (Capra Hircus), Homologous to Spermadhesins. Protein & Peptide Letters, 9, 331-335  https://doi.org/10.2174/0929866023408670
 
Teixeira DIA, Melo LM, Gadelha CAD, da Cunha RMS, Bloch Jr C, Radis-Baptista G, Cavada BS, Freitas VJD (2006): Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma. Genetics and Molecular Research 5, 79–87.
 
Thimon Véronique, Métayer Sonia, Belghazi Maya, Dacheux Françoise, Dacheux Jean-Louis, Gatti Jean-Luc (2005): Shedding of the Germinal Angiotensin I-Converting Enzyme (gACE) Involves a Serine Protease and Is Activated by Epididymal Fluid1. Biology of Reproduction, 73, 881-890  https://doi.org/10.1095/biolreprod.105.042929
 
Thuwanut P, Axnér E, Johanisson A, Chatdarong K (2009): Detection of Lipid Peroxidation Reaction in Frozen-Thawed Epididymal Cat Spermatozoa Using BODIPY 581/591 C11. Reproduction in Domestic Animals, 44, 373-376  https://doi.org/10.1111/j.1439-0531.2009.01453.x
 
Töpfer-Petersen E., Romero A., Varela P. F., Ekhlasi-Hundrieser M., Dostàlovà Z., Sanz L., Calvete J. J. (1998): Spermadhesins: A new protein family. Facts, hypotheses and perspectives. Andrologia, 30, 217-224  https://doi.org/10.1111/j.1439-0272.1998.tb01163.x
 
Troedsson M.H.T., Desvousges A., Alghamdi A.S., Dahms B., Dow C.A., Hayna J., Valesco R., Collahan P.T., Macpherson M.L., Pozor M., Buhi W.C. (2005): Components in seminal plasma regulating sperm transport and elimination. Animal Reproduction Science, 89, 171-186  https://doi.org/10.1016/j.anireprosci.2005.07.005
 
Tummaruk P., Lundeheim N., Einarsson S., Dalin A.-M. (2000): Reproductive Performance of Purebred Swedish Landrace and Swedish Yorkshire Sows: II. Effect of Mating Type, Weaning-to-first-service Interval and Lactation Length. Acta Agriculturae Scandinavica, Section A - Animal Science, 50, 217-224  https://doi.org/10.1080/090647000750014340
 
Villemure M, Lazure C, Manjunath P (2003): Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reproductive Biology and Endocrinology 1, doi: 10.1186/1477-7827-1-39. https://doi.org/10.1186/1477-7827-1-39
 
WHO – World Health Organization (1999): World Health Organization laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th edn. Cambridge University Press, Cambridge. 138 pp.
 
WHO – World Health Organization (2010): World Health Organization laboratory manual for the examination and processing of human semen. 5th edn. Cambridge University Press, Cambridge. Available at http://apps.who.int/iris/bitstream/handle/10665/44261/9789241547789_eng.pdf.
 
download PDF

© 2020 Czech Academy of Agricultural Sciences