Background and Aim: Protamine (PRM) is the major protein in the sperm nucleus and plays an essential role in its normal function. Moreover, PRM has great potential as a protein marker of semen production and quality. This study aimed to assess the potential of sperm bovine PRM as a protein marker of semen production and quality in bulls at the National Artificial Insemination (AI) Center of Indonesia. Materials and Methods: The semen production capacity of each bull was collected from frozen semen production data at the Singosari AI Center for 6 months, and was then divided into two groups (high and low). A total of 440 frozen semen straws from six Limousin (LIM), six Friesian Holstein (FH), six Peranakan Ongole (PO), and four Aceh bulls aged 4-5 years were used in the study. The frozen semen was used to measure the concentration of PRM1, PRM2, and PRM3 using the enzyme immunoassay method. The frozen semen was also used to assess the quality of the semen, including progressive motility (PM) through computer-assisted semen analysis, sperm viability through eosin–nigrosin analysis, and the DNA fragmentation index through Acridine Orange staining. Results: PRM1 was significantly higher in all bull breeds included in the study (p<0.00), followed by PRM2 (p<0.00) and PRM3 (p<0.00). PRM1 significantly affected semen production in LIM, FH, PO, and Aceh bulls (p<0.05). Moreover, PRM2 significantly affected semen production only in FH and Aceh bulls (p<0.05), whereas PRM3 affected this parameter in PO and Aceh bulls exclusively (p<0.05). Consistently and significantly, PRM1 was positively correlated with the PM and viability of sperm and negatively associated with its DNA fragmentation in LIM, FH, PO, and Aceh bulls (p<0.05; p<0.01). The correlation analysis between PRM2 and PRM3 and semen quality parameters varied across all bull breeds; some were positively and negatively correlated (p<0.05; p<0.01), and some were not correlated at all. Conclusion: PRM1 has excellent potential as a protein marker of semen production and quality in bulls at the National AI Center of Indonesia.

No abstract

Background and Aim: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques. Materials and Methods: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. Results: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05). Conclusion: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.

Abstrak. Kaiin EM, Gunawan M, Octaviana S, Nuswantara S. 2017. Verifikasi molekuler metode sexing sperma sapi dengan kolom BSA (Bovine Serum Albumin). Pros Sem Nas Masy Biodiv Indon 3: [241][242][243][244][245]. Penentuan jenis kelamin anak sapi merupakan salah satu langkah strategis dalam perkembangan teknologi inseminasi buatan. Metoda pemisahan spermatozoa pembawa kromosom jenis kelamin betina (X) atau jantan (Y) dengan kolom BSA telah menghasilkan keberhasilan kesesuaian jenis kelamin anak sapi yang di lapangan sebesar 76% sampai 89%. Tujuan dari penelitian ini ialah memverifikasi hasil pemisahan sperma pembawa kromosom X dan kromosom Y dengan metode kolom BSA (Bovine Serum Albumine) secara molekuler. Sperma sexing sapi Simmental yang telah dipisahkan kromosom X dan Y dengan kolom BSA 5% (sperma X) dan 10% (sperma Y), diuji kualitas sperma secara mikroskopis meliputi parameter motilitas, viabilitas dan abnormalitas sperma. DNA sperma pada masing-masing kolom (5% atau 10%) diekstrasi menggunakan metoda spin-column dan kemudian diamplifikasi menggunakan Polymerase Chain Reaction (PCR) dengan design primer gen Sex-determining Region Y (SRY) yang berada pada daerah kromosom Y dan gen autosomal GADPH (Glyceraldehyde 3-phosphate dehydrogenase) pada daerah HMG Box (High Mobility Group Box). Hasil penelitian menunjukkan bahwa motilitas sperma X dan Y sebesar 60-70%, viabilitas sperma X sebesar 76,9-80,1% dan sperma Y sebesar 75,5-77,7%, sedangkan abnormalitas sperma X sebesar 6,4-7,6% dan sperma Y sebesar 5,2-5,5%. Hasil pemisahan sperma pada kolom BSA konsentrasi 10% dan sperma yang tidak disexing (kontrol), terverifikasi adanya 2 pita yaitu gen SRY (318 bp) dan GAPDH (415 bp). Hal ini menunjukkan bahwa pada kolom BSA 10% terdapat lebih banyak sperma Y. Hasil pada kolom BSA 5%, hanya terdapat 1 pita GAPDH (415 bp), menunjukkan sperma pada kolom tersebut adalah sperma X. Hasil ini menunjukkan bahwa sexing sperma sapi dengan metoda kolom BSA 5% dan 10%, dapat terverifikasi secara molekuler memisahkan sperma sapi pembawa kromosom X dan Y yang diuji dengan menggunakan metode duplex PCR.Kata kunci: Sexing, sperma, BSA, PCR, SRY Abstract. Kaiin EM, Gunawan M, Octaviana S, Nuswantara S. 2017. Molecular verification of sperm sexing method with BSA (Bovine Serum Albumin) column. Pros Sem Nas Masy Biodiv Indon 3: 241-245.Determining the sex of cattle was a strategic step in the development of artificial insemination technology. Separation of sperm carriers female sex chromosome (X) or male sex chromosome (Y ) methods with a BSA column has produced successful sex-matched calves after Artificial Insemination (AI) by 76% to 89%. The aim of this study was to verify the results of sperm separation with a BSA column method based on molecular technique. Simmental sperm was separated with 5% BSA column (X sperm) and 10% BSA column (Y sperm). Sperm quality was tested microscopically with parameters: motility, viability, sperm abnormalities and intact membrane plasma (IMP). DNA of sperm from each column was extracted using spin-column...

Abstract. Kaiin EM, Gunawan M, Maulana T. 2017. Morphometry and abnormality evaluation of sex-sorted sperm of spotted buffalo (Tedong bonga). Nusantara Bioscience 9: 175-180. This research aimed to determine the morphometry and the abnormality of the spotted buffallo (Tedong bonga) sperm after the separation of X and Y-chromosome-bearing sperms. Semen was collected from the spotted buffalo bull by using an artificial vagina.The semen quality was evaluated macro-and microscopically. Semen that met the required standard were subjected to separation by using Bovine Serum Albumin (BSA) 5% and 10% column method. The quality of sex-sorted sperm was evaluated microscopically, including motility, concentration, viability, abnormality and intact plasma membrane. Smear preparation using Eosin-Nigrosin dyes was made for sperm morphometry and abnormality evaluation.The evaluation of sperm morphometry and abnormality was conducted on 200 sperm cells for each observation and repeated in triplicates of observation, by using Axiovision Imager Z microscope at objective and ocular magnifications 40x and 10x, respectively.The morphological parameters observed were including the length and the width of head sperm and the head area of sperm X or Y. The abnormality parameter consisted of primary and secondary abnormalities. The data were analyzed descriptively. The results showed that the head size of the Y sperm of spotted buffalo (27.16 µm 2 ) was smaller than that of the X sperm (29.86 µm 2 ) with the abnormality of the Y sperm (7.85%) was also lower than that of the X sperm (9.7%).