Comparison of Leja and Makler Chambers Performance in Rainbow Trout (Oncorhynchus mykiss) Sperm Quality in CASA System

Abstract

In this study, the effect of the chamber used for the automated analysis of sperm motility and sperm kinematics parameters by CASA was evaluated of rainbow trout (Oncorhynchus mykiss) sperms. The assessment of motility parameters was carried out using CEROS II (Hamilton-Thorne, Beverly, MA, USA) connected to CX41 microscope (Olympus, Japan) at room temperature. Sperm samples were collected from five adult males by abdominal massage during the reproduction season and analyzed with two different chambers as follows specialty: Leja 2 cell chambered with 20 µl deep (Leja Products, Netherlands) and Makler chamber, round shape with 10 µl deep (Sefi-Medical Instrument, Haifa, Israel). Total sperm motility (Mot, %), and Velocity of Curvilinear (µm/s) were measured. For fertilization test, eggs from one female (550 g approximately 7500 eggs) were separated in equal five parts. Each part of eggs (approximately 1.500 eggs) was fertilized with each analyzed sperm (5ml). Fertilization, incubation procedure and calculation of fertilization rates have been kept as used routinely for rainbow trout culture procedure. The fertilization rates were found ˃80% for all used males. The motility percentage of samples analyzed by Leja has been found higher 90% while by makler changed between 26-45%. There is significantly effect on different chambers used in this study to determining the motility percentage. The high stability results and matched the fertilization success were detected in Leja 2-chamber. Statistical study with motility percentage showed a significant difference between Leja and Makler chambers (p<0,05). 

Keywords

• CASA,Leja 2,Makler,Motility parameters,Fertilization rate,Rainbow trout

Gökkuşağı Alabalığının (Oncorhynchus mykiss) Sperm Kalitesinde Bilgisayarlı Otomatik Sperm Analiz Sisteminde (CASA) Leja ve Makler Lamlarının Performansının Karşılaştırılması

Öz

Bu çalışmada gökkuşağı alabalığının (Oncorhynchus mykiss) spermine ait motilite ve motiliteye ait kinematik parametrelerin Bilgisayarlı Otomatik Sperm Analiz Sistemi (CASA) ile incelenmesinde farklı lamların kullanılmasının sonuçlar üzerindeki etkisi araştırılmıştır. Motilite ve parametreleri, oda sıcaklığında CEROS II (Hamilton-Thorne, Beverly, MA, USA) yazılım sistemine bağlı ışık mikroskobu (CX41, Olympus, Japan) ile incelenmiştir. Sperm örnekleri beş erkek balıktan abdominal masaj yöntemi ile balıkların üreme döneminde toplanmış ve Leja 2 (20 µl deep, Leja Products, Netherlands) ve Makler (10 µl deep, Sefi-Medical Instrument, Haifa, Israel) olmak üzere iki farklı lam kullanılarak analiz edilmişlerdir. Sperm örneklerinde toplam motilite (%), ve hız parametrelerinden eğrisel hız olarak ifade edilen VCL (µm/s) analiz edilmiştir. Dölleme çalışması için, bir dişi balıktan alınan yumurtalar (toplam 550 g, 7500 yumurta) beş eşit parçaya bölünmüştür. Bölünen yumurtaların her bir bölümü (1500 yumurta) analize tabi tutulan sperm örnekleri (5 ml) ile döllenmiştir. Dölleme, inkübasyon ve döllenme oranı prosedürleri, alabalık yetiştiriciliği için kullanılan rutin uygulama prosedürü altında gerçekleştirilmiştir. Döllenme yüzdesi her yumurta grubu için ˃%80 olarak bulunmuştur. İncelenen sperm örneklerinde Makler lamın kullanılması ile sperm motilite değerleri %26-45 arasında değişiklik göstermişken; Leja 2 lamı kullanılarak incelenen sperm örneklerinde her bir balıkta motilite değeri %90 ve üzeri bulunmuştur. Analiz esnasında iki farklı lam kullanılmasının motilite sonuçlarının belirlenmesini etkilediği bulunmuştur. Motilite sonuçları ve dölleme sonuçları karşılaştırıldığında ise Leja 2 lamı ile incelenen örneklerin motilite sonuçları döllenme oranı ile örtüştüğü ve daha kararlı sonuçlar verdiği görülmüştür. Motilite sonuçları ile yapılan istatistiksel değerlendirmede Leja 2 ve Makler lamlarından elde edilen sonuçlar arasında anlamlı bir farklılık tespit edilmiştir (p<0.05).

Anahtar Kelimeler

• Leja 2,CASA,Makler,Motilite parametreleri,Döllenme oranı,Gökkuşağı alabalığı

Kaynakça

1. Bobe, J., & Labbé, C. (2010). Egg and sperm quality in fish. General and Comparative Endocrinology, 165(3), 535-548. DOI: 10.1016/j.ygcen.2009.02.011. Broekhuijse, M. L. W. J., Šoštarić, E., Feitsma, H., & Gadella, B. M. (2012). The value of microscopic semen motility assessment at collection for a commercial artificial insemination center, a retrospective study on factors explaining variation in fertility. Theriogenology, 77(7), 1466-1479.DOI: 10.1016/j.theriogenology.2011.11.016.
2. Bromage, NR., & Roberts, RJ. (1995). Broodstock Management and Egg and Larval Quality. Blackwell Science, Oxford.
3. Cabrita, E., Robles, V., & Herraez, P. (2008). Sperm Quality Assesment In: E. Cabrita, V. Robles P. Herraez (Ed). Methods in Reproductive Aquaculture, Marine and Fresh- water species. (pp 93-148). CRC Press, Taylor & Francis Group, Boca Raton, FL, USA.
4. Cabrita, E., Martínez-Páramo, S., Gavaia, P. J., Riesco, M. F., Valcarce, D. G., Sarasquete, C., & Robles, V. (2014). Factors enhancing fish sperm quality and emerging tools for sperm analysis. Aquaculture, 432, 389-401. DOI:10.1016/j.aquaculture.2014.04.034.
5. Caldeira, C., & Soler, C. (2018). Fish Sperm Assessment Using Software and Cooling Devices. Journal of Visualized Experiments, (137). DOI: 10.3791/56823.Castellini, C., Dal Bosco, A., Ruggeri, S., & Collodel, G. (2011). What is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis?. Fertility and Sterility, 96(1), 24-27. DOI: 10.1016/j.fertnstert.2011.04.096.
6. Chong, AP., Walters, CA., & Weinrieb, SA. (1983). The neglected laboratory test The semen analysis. Journal of Andrology, (4):280-2. DOI: 10.1002/j.1939-4640.1983.tb02368.x.
7. Christensen, P., Stryhn, H., & Hansen, C. (2005). Discrepancies in the determination of sperm concentration using Bürker-Türk, Thoma and Makler counting hambers. Theriogenology, 63(4), 992-1003.DOI: 10.1016/j.theriogenology.2004.05.026
8. Contri, A., Valorz, C., Faustini, M., Wegher, L., & Carluccio, A., 2010. Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa. Theriogenology, 74, 424–435. DOI: 10.1016/j.theriogenology.2010.02.025. Del Gallego, R., Sadeghi, S., Blasco, E., Soler, C., Yániz, J. L., & Silvestre, M. A. (2017). Effect of chamber characteristics, loading and analysis time on motility and kinetic variables analysed with the CASA-mot system in goat sperm. Animal Reproduction Science, 177, 97-104. DOI: 10.1016/j.anireprosci.2016.12.010.

9. Gączarzewicz, D. (2015). Influence of chamber type integrated with computer-assisted semen analysis (CASA) system on the results of boar semen evaluation. Polish Journal of Veterinary Sciences, 18(4), 817-824. DOI 10.1515/pjvs-2015-0106.
10. Gallego, V., & Asturiano, J. F. (2018). Sperm motility in fish: technical applications and perspectives through CASA-Mot systems. Reproduction, Fertility and Development, 30(6), 820-832. DOI: doi.org/10.1071/RD17460.
11. Gallego, V., & Asturiano, J. F. (2019). Fish sperm motility assessment as a tool for aquaculture research: a historical approach. Reviews in Aquaculture. 1-28. DOI: 10.1111/raq.12253.
12. Gloria, A., Carluccio, A., Contri, A., Wegher, L., Valorz, C., & Robbe, D. (2013). The effect of the chamber on kinetic results in cryopreserved bull spermatozoa. Andrology, 1(6), 879-885. DOI: 10.1111/j.2047-2927.2013.00121.x.
13. Hoogewijs, M. K., De Vliegher, S. P., Govaere, J. L., De Schauwer, C., de Kruif, A., & Van Soom, A. (2012). Influence of counting chamber type on CASA outcomes of equine semen analysis. Equine Veterinary Journal, 44(5), 542-549. DOI: 10.1111/j.2042-3306.2011.00523.x.
14. Ibănescu, I., Leiding, C., Ciornei, Ş. G., Roșca, P., Sfartz, I., & Drugociu, D. (2016). Differences in CASA output according to the chamber type when analyzing frozen-thawed bull sperm. Animal Reproduction Science, 166, 72-79. DOI: 10.1016/j.anireprosci.2016.01.005.
15. Iguer-Ouada, M., & Verstegen, J.P. (2001). Evaluation of the “Hamilton Thorn computer-based automated system” for dog semen analysis. Theriogenology, 55, 733–749. DOI: 10.1016/S0093-691X(01)00440-X.
16. Jasko, D. J., Leın, D. H., & Foote, R. H. (1990). A comparison of two computer‐automated semen analysis instruments for the evaluation of sperm motion characteristics in the stallion. Journal of Andrology, 11(5), 453-459. DOI: 10.1002/j.1939-4640.1990.tb00176.x.
17. Lenz, R. W., Kjelland, M. E., Vonderhaar, K., Swannack, T. M., & Moreno, J. F. (2011). A comparison of bovine seminal quality assessments using different viewing chambers with a computer-assisted semen analyzer. Journal of Animal Science, 89(2), 383-388. DOI: 10.2527/jas.2010-3056.
18. Mananos, E., Duncan, N., & Mylonas, C. (2008). Reproduction and control of ovulation, spermiation and spawning in cultured fish. In: E. Cabrita, V. Robles, P. Herraez P (Ed.) Methods in Reproductive Aquaculture, Marine and Fresh- water species (pp3-80). CRC Press, Taylor & Francis Group, Boca Raton, FL, USA.
19. Massányı, P. C., Žıvčak, J., & Bulla, J. (2008). Comparison of different evaluation chambers for analysis of rabbit spermatozoa motility parameters using CASA system. Slovak Journal of Animal Science, 41(2), 60-66.
20. Palacín, I., Vicente-Fiel, S., Santolaria, P., & Yániz, J. L. (2013). Standardization of CASA sperm motility assessment in the ram. Small Ruminant Research, 112(1-3), 128-135. DOI: 10.1016/j.smallrumres.2012.12.014.
21. Rijsselaere, T., Van Soom, A., Maes D & de Kruif A. (2003). Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer. Theriogenology, 60, 1553–1568. DOI: 10.1016/S0093-691X(03)00171-7.
22. Rurangwa, E., Kime, D. E., Ollevier, F., & Nash, J. P. (2004). The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture, 234(1-4), 1-28. DOI: 10.1016/j.aquaculture.2003.12.006.
23. Soler, C., del Carmen Fuentes, M., Sancho, M., Garcia, A., Nunez de Murga, M., & Nunez de Murga, J. (2012). Effect of counting chamber on seminal parameters, analyzing with the ISASv1 (R). Revista Internacional de Andrología, 10(4), 132-138.
24. Spizziri, B. E., Fox, M. H., Bruemmeer, J. E., Squires, E. L., & Graham, J. K. (2010). Cholesterol-loaded-cyclodextrins and the fertility potential of stallion spermatozoa. Animal Reproduction Science, 118, 255–264. DOI: 10.1016/j.anireprosci.2009.08.001.
25. Tomlinson, M. J., Pooley, K., Simpson, T., Newton, T., Hopkisson, J., Jayaprakasan, K., & Pridmore, T. (2010). Validation of a novel computer-assisted sperm analysis (CASA) system using multitarget-tracking algorithms. Fertility and Sterility, 93(6), 1911-1920. DOI: 10.1016/j.fertnstert.2008.12.064.
26. Verstegen, J., Iguer-Ouada M & Onclin K. (2002). Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology, 57, 149–179. DOI: 10.1016/S0093-691X(01)00664-1.