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.

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.

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.

ABSTRAKPenelitian ini bertujuan menganalisis perubahan ukuran kepala dan integritas DNA spermatozoa sapi kering beku. Sperma kering beku disimpan pada temperature 4°C selama 2 tahun. Ukuran kepala sperma ditentukan dengan menggunakan Computer-assisted sperm analysis (CASA), sedangkan integritas DNA dianalisis pewarnaan acridine orange. Preparat ulas spermatozoa dibuat dan difiksasi dengan acetic alcoholdandiwarnai dengan acridine orange. Setelah diwarnai, setiap slide diamati dibawah mikroskop fluoresens pembesaran 400X dengan axio vision (Zeiss Company, Germany). Hasil penelitian menunjukkan bahwa ukuran kepala sperma membesar signifikan (P<0,05) setelah dikeringbekukan, sebaliknya kepala sperma mengalami pengecilan ukuran secara signifikan (P<0,05) setelah diinkubasi selama 3 dan 6 jam. Integritas DNA spermatozoa kering beku berbeda nyata (P<0,05) menurun setelah inkubasi selama 6 jam. Dapat disimpulkan bahwa (1) kepala spermatozoa mengalami pembesaran setalah melalui proses pengeringbekuan, sebaliknya mengalami pengecilan setelah proses inkubasi, (2) integritas DNA spermatozoa kering beku tetap utuh selama inkubasi 3 jam dan penurunan integritas DNA terjadi setelah inkubasi selama 6 jam.Kata kunci : ukuran bentuk, integritas DNA, spermatozoa kering-beku, sapi, inkubasi ABSTRACTChanges of sperm heads morphometric and DNA integrity of freeze-dried bovine spermatozoa were investigated. Freeze-dried spermatozoa had stored in the refrigerator at 4°C for 2 years. Computerassisted sperm analysis (CASA) was used in this study to identify sperm head morphometry, while for DNA integrity analysis using acridine orange staining. Samples were smeared on glass slides, fixed for 2 h in acetic alcohol and stained with acridine orange solution. After staining, each slide was examined at x400 magnification in a fluorescence microscope with axio vision (Zeiss Company, Germany). Proportion of fluorescence red and green emissions of the sperm head were examined and scored. These results indicated that sperm head had enlarged significantly (P<0.05) after freeze-drying process. However, freeze-dried sperm heads morphometry significantly (P<0.05) decrease after incubation for 3 and 6 hours. Changes of DNA integrity of freeze-dried spermatozoa significantly (P<0.05) decrease after incubation for 6 hours. In the present study concluded that (1) freeze-drying spermatozoa caused sperm head morphometric enlarged, whereas incubation time caused sperm heads decreased, (2) DNA integrity of freeze-dried sperm head is still intact during incubation 3 hours, and decreased DNA integrity occur in incubation for 6 hours.

<p class="abstrak1"><span lang="IN">The objective of the current study was to asses the optimal concentration of glutamine, glycine and cysteine amino acids in tris-citric-acid-fructose egg yolks (TCFY) extender on quality of SO bull spermatozoa during freezing and thawing. </span><span lang="EN-US">In t</span><span lang="IN">his study the DNA stability of frozen-thawed Sperm</span><span lang="EN-US"> was </span><span lang="IN">also indentified. Three mature bulls maintained at PT. Karya Anugerah Rumpin, private cattle breeding company, West Java, Indonesia were used as semen donors. Semen was collected using artificial vagina and were evaluated </span><span lang="EN-US">prior</span><span lang="IN"> to freezing. Semen was diluted with TCFY supplemented with different concentrations of amino acids (5, 15 and 25 mM glycine and glutamine, and 3, 5 and 7 mM cysteine) then processed for colling and freezing. Semen quality parameters (subjective motility, viability and membrane and DNA integrity). </span><span lang="EN-US">D</span><span lang="IN">ata showed that in general the effect of addition of selected amino acids (glycine, glutamine and cysteine) </span><span lang="EN-US">in</span><span lang="IN">to TCFY extenders on motility, viability and membrane integrity of SO spermatozoa after cooling were significantly different (p&lt;0.05) higher than</span><span lang="EN-US"> that of</span><span lang="IN"> control. Addition of 15 mM glycine, 15 mM glutamine and 5 mM cysteine resulted in significant (p&lt;0.05) increase post-thawing sperm motility and sperm viability as compared to th</span><span lang="EN-US">at of</span><span lang="IN"> control. Furthermore, when spermatozoa were stained with acridine orange after fixation with acetic alcohol, the DNA integrity of post-thawing spermatozoa showed that all spermatozoa were remain intact. In conclusion </span><span lang="EN-US">,</span><span lang="IN">addition of 15 mM glycine, glutamine and 5 mM cysteine increase the cryoprotecting efficacy of bovine bull cryopreservation extender, and furthermore all DNA spermatozoa were remain intact. </span></p>

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%).

This study was conducted to determine the quality fresh and frozen semen of Bali and Simmental bull in the Technical Implementation Unit of Regional Artificial Insemination Center at Pucak, South Sulawesi. Two Bali bulls and two Simmental bulls aged 5-6 were used. The parameters observed in the fresh semen were volume, pH, colour, motility, viability, abnormality, intact plasma membrane, and concentration. Meanwhile, parameters observed in frozen semen were motility, viability, abnormality, intact plasma membrane, acrosome status, DNA integrity, and movement patterns. Viability and abnormality parameters were stained with eosin-nigrosine, intact plasma membrane was stained with Hypo-osmotic (HOS) solution, acrosome status was stained with peanut agglutinin (PNA), and DNA integrity was stained with acridine orange (AO). The results showed that the volume, pH, motility, abnormality, intact plasma membrane, and concentration of fresh semen of Bali and Simmental bull were not significantly different (P>0.05). Likewise, the frozen semen of Bali and Simmental breed did not show any significant differences (P>0.05) in terms of motility, acrosome status, intact plasma membrane, DNA integrity, and movement patterns, but the viability of Bali bull was significantly higher than that of Simmental bull and the abnormality of Simmental bull was significantly higher than that of Bali bull. Based on the results, it can be concluded both Bali and Simmental bull has the same quality of fresh semen, but Bali bull had better viability and abnormality compared to those Simmental breed of frozen semen.

The study aimed to examine the kinematics motility and recovery rates of frozen-thawed Friesian Holstein bull sexing semen in the addition of l-ascorbic acid treatment. Semen collected from Friesian Holstein bulls with the minimum standard of sperm motilities for sexing sperm (60%), and abnormality (<20%) used for this study. Semen was diluted with final concentration 300x106 sperm/ml in Brackett-Oliphant (BO) medium. The sperms were sorted using the BSA column method. Sexing sperm were extended with Tris egg yolk (TEY) extender in addition L-Ascorbic Acid (LA) 0%, 0.25%, 0.5%, and 0.75% before freezing process. Frozen sexing semen was thawed for the kinematics motility evaluation with parameter; Tmot, Pmot, VAP, VCL, VSL, LIN, ALH, BCF, DAP, DCL and DAP using CASA system also recovery-rate evaluation. The results showed kinematics motility evaluation for 0.75% TEY-LA had lowest value (P<0.05) in Tmot, Pmot, velocity and distance compared to control group, 0.25% and 0.5% TEY-LA. The recovery rate 0.5% TEY-LA had highest values between among all treatment in X and Y frozen sexing semen. In conclusion, the addition of 0.5% l-ascorbic acid to tris egg yolk extender could be advantageous tool to improve the kinematics motility and increased recovery rates of Friesian Holstein sexing sperm.