Sains Malaysiana 44(9)(2015): 1283–1288

Effect of Different Cryoprotectants and Sperm Densities of Orange Mud Crab, Scylla olivacea (Herbst, 1796) for Long-Term Storage of Spermatozoa

(Kesan Perbezaan Kepadatan Sperma dan Kryoprotektan ke atas Penyimpanan Jangka Panjang Spermatozoa Ketam Bakau, Scylla olivacea (Herbst, 1796))

MHD IKHWANUDDIN¹*, TENG PHEI YIN², ABDUL JABBAR MENON³, SAFIAH JASMANI¹ & AMBOK BOLONG ABOL-MUNAF⁴

¹Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, Malaysia

²School of Marine Science and Environment, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, Malaysia

³Directorate of Fisheries Inland Hyderabad, Live Stock & Fisheries Department, Government of Sindh, Pakistan

⁴School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, Malaysia

Received: 12 August 2014/Accepted: 26 May 2015

ABSTRACT

The objectives of this study were to determine the effect of different cryoprotectants and sperm densities for long-term storage of orange mud crab, Scylla olivacea spermatozoa. Spermatozoa were obtained by homogenizing the spermatophores using a glass homogenizer in an ice-bath followed by centrifugation at 4°C. Spermatozoa were then suspended in calcium-free saline (Ca-F saline) containing 5% of the following cryoprotectants: Glycerol, dimethyl sulfoxide (DMSO) and methanol. Sperm which vibrated and rotated were counted as live during sperm viability assessment. Samples of spermatozoa were cooled to -196°C by two-step freezing, first to -80°C and then by plunging into liquid nitrogen (LN). Spermatozoa were gradually cooled at 1°C/min. Thawing was carried out in a 30°C water bath for 2 min. This yielded live sperm after storage in LN for 30 days. The best sperm viability was obtained from a density of 108 cells per mL in DMSO. There was no significant difference (p>0.05) among cryoprotectants toward sperm viability. However, sperm viability was significantly affected (p>0.05) by cell densities. In conclusion, DMSO gave the best protection to sperm cells of S. olivacea, but the effectiveness of DMSO as a cryoprotectant is influenced by sperm density.

Keywords: Cryprotectants; orange mud crab; Scylla olivacea; sperm density; spermatozoa

ABSTRAK

Objektif kajian ini adalah untuk menentukan kesan krioprotektan dan kepadatan sperma untuk penyimpanan jangka panjang spermatozoa ketam bakau, Scylla olivacea. Spermatozoa diperoleh daripada proses penghomogenan spermatofor menggunakan kaca penghomogenan di dalam takungan yang berisi ais dan diikuti dengan proses penapisan pada suhu 4ºC. Kemudian, spermatozoa dicampurkan dengan salin bebas-kalsium (Ca-F saline) yang mengandungi gliserol krayoprotektan, dimetil sulfoksida (DMSO) dan metanol masing-masing pada kepekatan 5%. Sperma yang bergegar dan berputar akan dikira sebagai sperma yang hidup di dalam penilaian kemajuan sperma. Sampel spermatozoa kemudiannya disejukkan pada suhu -196°C melalui dua langkah pembekuan, pertama pada suhu -80°C dan kemudian dimasukkan ke dalam cecair nitrogen (LN). Penyejukkan secara beransur-ansur pada 1°C/min telah dijalankan dengan menyejukkan spermatozoa tersebut. Pencairan dilakukan pada suhu 30°C di dalam bekas takungan berisi air selama 2 min. Ini menghasilkan sperma yang hidup di dalam simpanan LN selama 30 hari. Kemajuan sperma yang terbaik diperoleh daripada kepadatan 108 sel per mL di dalam DMSO. Tiada perbezaan yang ketara (p>0.05) antara krioprotektan dan kemajuan sperma. Walau bagaimanapun, terdapat perbezaan yang ketara (p<0.05) antara kemajuan sperma dan kepadatan selnya. Kesimpulannya, DMSO memberikan perlindungan yang terbaik ke atas sel sperma S. olivacea, tetapi keberkesanan DMSO sebagai krayoprotektan dipengaruhi oleh ketumpatan sperma yang digunakan.

Kata kunci: Kepadatan sperma; ketam bakau; krioprotektan; Scylla olivacea; spermatozoa

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*Corresponding author; email: ikhwanuddin@umt.edu.my