Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission.

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ABSTRAKTujuan dari penelitian ini adalah untuk menguji efektivitas dari tiga macam bahan pengencer (Tris kuning telur, Bioxcell® dan Optixcell®) dan tiga waktu ekuilibrasi (T-30 min, T-2 jam dan T-4 jam) untuk kriopreservasi semen sapi bali. Tiga puluh enam ejakulat semen berasal dari 6 ekor sapi bali jantan dikoleksi dan dibekukan mengikuti protokol pembekuan rutin. Evaluasi motilitas sperma postthawing (PTM) dilakukan dengan computer assisted semen analysis (CASA). Integritas membran plasma dievaluasi dengan hypo-osmotic swelling (HOS) test dan viabilitas dievaluasi dengan pewarnaan eosin nigrosin. Hasil penelitian menunjukkan terdapat interaksi yang signifikan antara waktu ekuilibrasi dan pengencer terhadap motilitas, viabilitas dan integritas membran sperma sapi bali. Perlakuan kontrol (4 jam waktu ekuilibrasi) memiliki nilai tertinggi (P<0,05) dan perlakuan 30 menit waktu ekuilibrasi memiliki nilai terrendah (P<0,05) untuk total motilitas, motilitas progresif, persentase sperma dengan membran plasma utuh (MPU) dan viabilitas sperma. Motilitas, skor pergerakan individu dan integritas membran sperma dalam pengencer Optixcell® lebih tinggi dibandingkan dengan semen yang dibekukan dalam pengencer Bioxcell® dan Tris kuning telur. Motilitas total, progresif dan integritas membran sperma terbaik secara keseluruhan terdapat dalam pengencer Tris kuning telur dan Bioxcell® dengan waktu ekuilibrasi 4 jam dan Optixcell® dengan waktu ekuilibrasi 2 dan 4 jam. Kesimpulan berdasarkan analisis subyektif dan obyektif, equilibrasi 30 menit tidak cukup untuk mempertahankan motilitas dan integritas membran sperma yang lebih baik; equilibrasi untuk 2 jam menghasilkan motilitas sperma tertinggi untuk pengencer Optixcell®; dan equilibrasi untuk 4 jam menghasilkan viabilitas tertinggi untuk semua pengencer yang digunakan.Kata kunci: sapi bali, kriopreservasi semen, pengencer semen, waktu ekuilibrasi, CASA. ABSTRACTThe objectives of the present study were to compare and determine the best post-thawed characteristics of balinese bull sperm cryopreserved in three different extenders; animal based (Trisclarified egg yolk (Tris-cEY)), and non-animal based extenders (Bioxcell® (lecithin based) andCharacteristics of Post-thawed Bali Bull Semen (A.S. Amal et al.) 135 Optixcell® (liposome based)) in combination with three different equilibration times (30 minutes, 2 hours, 4hours). Thirty six ejaculates were collected from six Balinese bulls and frozen in three extenders (Tris-cEY, Bioxcell® and Optixcell®) after equilibration in three different times (30 minutes, 2hours and 4hours). Computer-assisted sperm analysis (CASA), hypo-osmotic swelling test (HOST) and eosin nigrosin staining were used in the post-thawed semen analysis. There was a significant interaction between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P<0.05) for all the evaluated parameters independent of extender type. Overall, semen extended in Tris-cEY, Bioxcell® and O...

This study was conducted to investigate the effects of sericin on meiotic maturation, hydrogen peroxide (H 2 O 2) concentration and deoxyribonucleic acid (DNA) fragmentation in buffalo oocytes. Oocytes were matured in vitro in tissue culture medium (TCM-199) with in vitro maturation (IVM) groups under several conditions, namely without bovine serum albumin (-BSA), (+BSA), and 0.025%, 0.05%, 0.1% and 0.25% (w/v) sericin. The results showed that supplementation of the maturation medium with 0.05% sericin significantly increased the rate of oocytes that reach metaphase II compared with other groups, except for the 0.025% sericin-treated group. Intracellular H 2 O 2 concentrations in oocytes of all groups were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA). The concentration of H 2 O 2 in matured oocytes treated with 0.05% sericin was lower than in other groups. DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP digoxigenin nick end-labelling (TUNEL) method. The total proportion of TUNEL-positive oocytes at the MII stage was lower in the 0.05% sericin group. The results indicate that addition of 0.05% sericin to the maturation medium may improve nuclear maturation and may inhibit DNA fragmentation in oocytes by decreasing H 2 O 2 concentrations.

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ABSTRAK Penelitian ini dilakukan untuk mengevaluasi penggunaan metode swim up untuk persiapan sperma-tozoa sebelum fertilisasi terhadap tingkat fertilisasi in vitro spermatozoa kauda epididimis pasca pe-nyimpanan selama 48 jam. Kauda epididimis domba disimpan pada suhu 4 o C selama 0 hari (H-0), 1 hari (H-1) dan 2 hari (H-2), kemudian spermatozoa dikoleksi dan dibekukan. Spermatozoa ejakulat beku di-gunakan sebagai kontrol. Oosit yang telah matang difertilisasi secara in vitro dengan spermatozoa asal kauda epididimis pasca penyimpanan dan ejakulat menggunakan metode persiapan spermatozoa de-ngan dan tanpa swim up. Hasil penelitian menunjukkan bahwa spermatozoa asal kauda epididimis yang dikoleksi segera setelah kematian hewan (H-0) memiliki kemampuan yang sama dengan spermatozoa ejakulat (P>0,05). Tingkat fertilisasi spermatozoa kauda epididimis pasca penyimpanan selama 2 hari mengalami penurunan seiring bertambahnya waktu simpan. Penggunaan metode swim up dan tanpa swim up menunjukkan kemampuan fertilisasi yang sama pada spermatozoa ejakulat dan spermatozoa kauda epididimis yang disimpan. Dapat disimpulkan bahwa metode swim up tidak berpengaruh terha-dap tingkat fertilisasi in vitro spermatozoa asal kauda epididimis yang disimpan pada suhu 4 o C selama 2 hari. Kemampuan fertilisasi spermatozoa asal kauda epididimis domba yang disimpan pada suhu 4 °C mengalami penurunan sampai hari kedua, namun spermatozoa tersebut masih mampu membuahi oosit secara in vitro. ABSTRACT This aim of the present study was to evaluate the using of swim up method for sperm preparation before in vitro fertilization of epididymidal spermatozoa after storage for 48h. Epididymides were stored at 4 °C for 0 day (H-0), 1 day (H-1) and 2 days (H-2), afterward semen were collected and frozen. Ejaculated semen was used as control group. Matured oocytes were then in vitro fertilized by frozen-thawed spermatozoa of each group experiments and ejaculated semen using preparation methods with and without swim up. These results show in vitro fertilization ability of cauda epididymal ram sper-matozoa were collected immediately after the animal's death (H-0) is similar to ejaculated spermatozoa (P>0.05). The fertilizing ability of cauda epididymal after storage for 2 days was declined gradually of increased storage time. There was similar ability in fertilization rate of stored cauda epididymal sperma-tozoa between with or without swim up method (P>0.05). It can be concluded that method of swim up and without swim up were similar effect on the rate of in vitro fertilization epididymidal spermatozoa that stored at 4 °C for 2 days. The fertilizing ability spermatozoa collected from cauda epididymides were stored at 4 °C gradually decreased as the storage period was prolonged, however it still able to be fertilize oocytes in vitro.

The objective of this research was to investigate the potential of snakehead albumin extract (channalbumin) for sorting X and Y sperm of Sumba Ongole (SO) and its characteristic. Semen was collected from three SO bulls using artificial vagina and the freeze dried channalbumin was extracted from snakehead fish. Channalbumin column was made with different concentration ratio of top and bottom fraction: 2%:4%; 3%:5%; 4%:6% respectively and BSA 5%:10% as control. Semen was put in top fraction then incubated for 30 min at room temperature then each fraction was centrifuged at 1800 rpm for 10 minutes. The pellet was evaluated for motility, abnormality, viability, membrane integrity and head sperm morphometric. The results showed that the channalbumin capable to maintain sperm motility in the top fraction better than the bottom fraction. Sperm viability and membrane integrity in control group were significantly higher (P<0.05) than all channalbumin treatment. BSA 5%:10% has highest proportion of X and Y sperm (69%:76.77%) compared with 2%:4% (42.33%:79.13%), 3%:5% (55.97%:75.73%) and 4%:6% of channalbumin (62.77%:68%). It’s concluded that channalbumin 4%: 6% was effective for separation of XY sperm with higher proportion.

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