Disertasi

Efek kombinasi gliserol dan rafinosa pada kriopreservasi spermatozoa mencit terhadap membran plasma, membran mitokondria, ultrastruktur, Caspase-3, HSPA1 dan kualitas embrio = Effect combination of glycerol and raffinose on cryopreservation of mice spermatozoa on plasma membrane, mitochondrial membrane, ultrastructure, Caspase-3, HSPA1 and embryo quality.

Kriopreservasi spermatozoa merupakan proses integral dari sebagian besar teknologi reproduksi berbantu, seperti inseminasi buatan dan fertilisasi in-vitro. Namun, kriopreservasi sering mengakibatkan penurunan kualitas spermatozoa post-thawing dan cryoinjury. Tujuan penelitian ini adalah untuk menganalisis efek kriopreservasi spermatozoa menggunakan modifikasi krioprotektan (gliserol, rafinosa) versus Nakagata (rafinosa) atau Kitazato (gliserol, trehalosa) terhadap kualitas embrio. Sampel dikumpulkan dari 48 ekor mencit jantan dan betina Mus musculus albinus strain DDY yang di nekropsi dan spermatozoa diambil dari kauda epididimis. Sedangkan oosit mencit betina dikoleksi setelah dilakukan superovulasi dengan PMSG dan hCG. Kemudian dibagi menjadi empat kelompok yaitu spermatozoa segar (kontrol), spermatozoa bekucair menggunakan krioprotektan Nakagata, modifikasi krioprotektan dan krioprotektan Kitazato. Kualitas spermatozoa diamati berdasarkan konsentrasi, motilitas, morfologi, cryo-survival rate, viability rate dan integritas membran plasma. Embrio diperoleh melalui fertilisasi in-vitro konvensional antara kelompok kontrol dengan kelompok perlakuan menggunakan oosit segar. Kemudian perkembangan embrio diamati pada hari pertama dan ketiga berdasarkan jumlah dan similaritas blastomer serta presentase fragmentasi sel. Sedangkan ekspresi protein Caspase-3, HSPA1, ultrastruktur spermatozoa dan potensial membran mitokondria, berturut-turut dideteksi menggunakan imunositokimia, Western blot, Scanning electron micsroscope dan JC-10 kit. Data yang diperoleh kemudian dibandingkan antara sebelum dan setelah vitrifikasi serta antar kelompok perlakuan. Kriopreservasi spermatozoa menunjukkan pengaruh negatif terhadap kualitas embrio. Rendahnya kualitas embrio berkaitan dengan Cryosurvival Rate, Viability Rate, morfologi, integritas membran plasma, ekspresi protein HSPA1, ultrastruktur spermatozoa, dan potensial membran mitokondria. Namun, tidak ada perbedaan bermakna pada semua kelompok perlakuan berdasarkan ekspresi protein Caspase-3, HSPA1, ultrastruktur spermatozoa, potensial membran mitokondria, dan kualitas embrio. Modifikasi krioprotektan dapat menjadi alternatif pada kriopreservasi spermatozoa mencit melalui fertilisasi in vitro.
Kata Kunci : gliserol, rafinosa, trehalosa, kriopreservasi spermatozoa, kualitas embrio.


Cryopreservation of spermatozoa is an integral process of most assisted reproductive technologies, such as artificial insemination and in-vitro fertilization. However, cryopreservation often results in decreased spermatozoa quality post-thawing and cryoinjury. This study aimed to analyze the effect of spermatozoa cryopreservation by using modified cryoprotectant (glycerol, raffinose), versus Nakagata (raffinose) or Kitazato (glycerol, trehalose) on embryo quality. Samples were collected from 48 male and female Mus musculus albinus strain DDY mice which were necropsied and sperm taken from the cauda epididymis. Meanwhile, oocytes from female mice were collected after superovulation with PMSG and hCG. Then divided into four groups, namely fresh spermatozoa (control), frozen-thawed spermatozoa using Nakagata cryoprotectant, modified cryoprotectant, and Kitazato cryoprotectant. The spermatozoa quality is observed based on the concentration, motility, morphology, cryo-survival rate, viability rate, and plasma membrane integrity. Embryos were obtained through conventional invitro fertilization between the control group and cryoprotectant treatment with fresh oocytes. Then the development of the embryo is observed on the first and third days based on the number of blastomeres, equal of blastomeres, and cell fragmentation. Meanwhile, the protein expression of Caspase-3, HSPA1, spermatozoa ultrastructure, and mitochondrial membrane potential were respectively detected using immunocytochemistry, Western blot, scanning electron microscope, and JC-10 kit. Then, the data obtained was compared before and after vitrification. Cryopreservation of spermatozoa showed negative influence on embryo quality. Low embryo quality is related to Cryosurvival Rate, Viability Rate, morphology, plasma membrane integrity, HSPA1 protein expression, spermatozoa ultrastructure, and mitochondrial membrane potential. However, there were no significant differences in all treatment groups based on protein expression of Caspase-3, HSPA1, spermatozoa ultrastructure, mitochondrial membrane potential, and embryo quality. In summary, modified cryoprotectant can be an alternative for cryopreserving mice spermatozoa through in vitro fertilization.
Keywords: glycerol, raffinose, trehalose, cryopreservation of spermatozoa, embryo quality.