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1
Seidel, G. E. "Overview of sexing sperm." Theriogenology 68, no. 3 (August 2007): 443–46. http://dx.doi.org/10.1016/j.theriogenology.2007.04.005.
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Seidel, G. E., and L. A. Johnson. "Sexing mammalian sperm — Overview." Theriogenology 52, no. 8 (December 1999): 1267–72. http://dx.doi.org/10.1016/s0093-691x(99)00215-0.
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Sharma, Mridula, and Nishant Sharma. "Sperm Sexing in Animals." Advances in Animal and Veterinary Sciences 4, no. 10 (2016): 543–49. http://dx.doi.org/10.14737/journal.aavs/2016/4.10.543.549.
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Salinas, Paulo. "Flow Cytometry and Sperm Sexing in Animals." International Journal of Medical and Surgical Sciences 3, no. 3 (October 26, 2018): 893–902. http://dx.doi.org/10.32457/ijmss.2016.022.
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Flow cytometry is a useful technology in the sexed sperm, which measures and analyzes simultaneously, multiple physical characteristics of the cell, as they flow in a stream flow, through a light beam. The measured properties are the size of a particle, relative internal granularity, relative complexity and relative fluorescence intensity. Currently, hundreds of calves have been gestated through artificial insemination with sexed sperm in animal production. Since 1992, flow cytometry has been used, a technique that allows spermatozoa X and Y differentiation by DNA content. There is no other practical technique for sperm sexing to keep sperm functionality. The objectives of this review are to explain: (1) why the sperm containing the X or Y chromosome are phenotypically similar, but differ among themselves, (2) the principles and procedures used for sexing sperm by flow cytometry and sorting ( 3) accuracy, speed and efficiency of current procedure sperm sexing, (4) sperm damage occurred during sperm sexing and consequently the effects on fertility.
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Espinosa-Cervantes, Román, and Alejandro Córdova-Izquierdo. "Sexing sperm of domestic animals." Tropical Animal Health and Production 45, no. 1 (July 25, 2012): 1–8. http://dx.doi.org/10.1007/s11250-012-0215-0.
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Lele, Yanpiterson Umbu, Enike Dwi Kusumawati, and Aju Tjatur Nugroho Krisnaningsih. "Motilitas dan viabilitas spermatozoa semen sexing kambing peranakan etawa (pe) menggunakan metode sedimentasi putih telur dengan pengencer yang berbeda." Jurnal Sains Peternakan 5, no. 1 (June 1, 2017): 50–56. http://dx.doi.org/10.21067/jsp.v5i1.3140.
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ABSTRAK
Tujuan penelitian ini adalah untuk mengetahui motilitas dan viabilitas spermatozoa semen hasil sexing kambing Peranakan Etawa (PE) dengan metode sedimentasi putih telur menggunakan pengencer yang berbeda. Materi yang digunakan dalam penelitian ini adalah semen segar kambing Peranakan Etawa (PE) berumur 2 tahun dan bobot badan 120 kg dari Balai Besar Inseminasi Buatan (BBIB) Singosari Malang. Metode penelitian ini yang digunakan adalah penelitian laboratorium dengan menggunakan rancangan acak lengkap (RAL). Perlakuan terdiri dari semen sexing dengan menggunakan pengencer Andromed lapisan atas dan Andromed lapisan bawah dan Tris Aminomethan Kuning Telur lapisan atas dan Tris Aminomethan Kuning Telur lapisan bawah dengan masing-masing 10 ulangan. Variabel yang diamati adalah motilitas dan viabilitas spermatozoa semen sexing dengan pengencer yang berbeda, proses sexing menggunakan metode sedimentasi putih telur. Data yang diperoleh dianalisis menggunakan Analisis Varian (ANOVA) apabila perlakuan memberikan perbedaan, maka dilanjutkan dengan Uji BNT. Hasil penelitian menunjukkan bahwa metode sedimentasi putih telur dengan pengencer yang berbeda pada kambing Peranakan Etawa (PE) memberikan pengaruh yang sangat nyata (P<0,01) terhadap motilitas dan viabilitas spermatozoa semen sexing. Motilitas dan viabilitas terbaik terdapat pada pengencer Tris aminomethan kuning telur lapisan atas sebesar 75,65%, 74,41% dan Andromed lapisan atas sebesar 65,4%, 60%. Berdasarkan hasil penelitian dapat disimpulkan bahwa kualitas spermatozoa semen sexing kambing PE dengan menggunakan pengencer Tris aminomethan memberikan hasil terbaik terhadap motilitas dan viabilitas. Berdasarkan hasil penelitian ini maka disarankan agar menggunakan pengencer Tris aminomethan kuning telur sebagai pengencer sexing spermatozoa.
ABSTRACT
The purpose of this study was to determine the motility and viability of PE sperm sexing with egg white sedimentation method using different diluents. This research method used was laboratory research using a completely randomized design (CRD) treatment PE sexing sperm. The results showed that the quality of PE sperm goat with various diluents showed a significant influence (P<0,01). The variables observed were motility and viability of sperm. The data obtained were analyzed using variance analysis (ANOVA) with complete randomized design (CRD). Motility and viability at best against Tris aminomethane top layer of egg yolk 65,4% and the top layer of Andromed 75,65%, 74,41%. Based on the results it is suggested that using Tris aminomethane yolk as a diluents sexing sperm. It can be concluded that the quality of sexing sperm goat PE by using tris aminomethane diluent gives the best result on motility and viability.
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Feng, Y. L., L. Ma, J. L. McKeeby, B. Y. Chen, and J. L. Hall. "Sexing Individual Sperm Cells During Intracytoplasmic Sperm Injection (ICSI)." Fertility and Sterility 74, no. 3 (September 2000): S18—S19. http://dx.doi.org/10.1016/s0015-0282(00)00774-3.
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Garner, D. L., and G. E. Seidel Jr. "Past, present and future perspectives on sexing sperm." Canadian Journal of Animal Science 83, no. 3 (September 1, 2003): 375–84. http://dx.doi.org/10.4141/a03-022.
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Development of flow cytometry for sorting mammalian sperm according to their sex chromosomes began in the late 1970s and early 1980s. This technology, which has recently been commercialized for bovine sperm, is based on the differences in DNA content between X- and Y-chromosome-bearing sperm. Under ideal conditions, 5000 live bovine sperm of each sex can be sorted per second at 90% accuracy. Pregnancy rates of 50% have been achieved routinely in well-managed heifers with sex-sorted, cryopreserved bovine sperm compared to 60–80% with unsexed control sperm. About 90% of offspring have been of the selected sex. Sorting sperm according to sex chromosome content is similarly successful in many other mammals including exotic species, but sorting efficiencies are somewhat less for sperm from some species. Key words: Mammals, sex chromosomes, flow cytometer, cell sorter, DNA content, X and Y sperm, Hoechst 33342
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Garner, Duane L. "Flow cytometric sexing of mammalian sperm." Theriogenology 65, no. 5 (March 2006): 943–57. http://dx.doi.org/10.1016/j.theriogenology.2005.09.009.
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Morrell, J., K. Keeler, D. Noakes, N. Mackenzie, and D. Dresser. "Sexing of sperm by flow cytometry." Veterinary Record 122, no. 14 (April 2, 1988): 322–24. http://dx.doi.org/10.1136/vr.122.14.322.
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Hayakawa, Hiroyuki. "Sperm Sexing in the Cattle Industry." Journal of Mammalian Ova Research 29, no. 3 (October 2012): 119–23. http://dx.doi.org/10.1274/jmor.29.119.
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Bochenek, M., and Z. Smorag. "258 THE EFFECT OF A PLANT PROTEIN COMPONENT OF MEDIA USED FOR BULL SPERM SEXING ON SPERM MEMBRANE STATUS." Reproduction, Fertility and Development 20, no. 1 (2008): 209. http://dx.doi.org/10.1071/rdv20n1ab258.
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The aim of the work was to examine the effect of modified TALP medium (TALP/Pp, Animal Pharma B.V., Hengelo, The Netherlands)—used in the sperm sexing procedure—on bull sperm membrane status. The TALP was modified by replacement of bovine serum albumin (BSA) with a mixture of several plant proteins and soya lecithin (Pp). The Pp component was prepared using a high pressure homogenization process. The TALP/Pp had the same pH and osmotic pressure as the original TALP medium (TALP/BSA). The work was divided into 2 parts: (1) Nine ejaculates collected from 2 bulls (Holstein and Polish Red) were used. Immediately after collection, each ejaculate was split into 2 parts and diluted (1:2) with TALP/BSA or TALP/Pp. The sperm membrane status was examined after 3 days of storage at 15�C. (2) Fifteen ejaculates collected from 5 bulls (Holstein, Polish Red, and Simmental) were used. Each ejaculate was split into 2 parts: the first part was diluted with TALP/BSA, stained, incubated, and sexed according to the XY Inc. bull semen sexing procedure; the second part was diluted, stained, incubated, and collected after sexing into TALP/Pp with no egg yolk addition. In both groups no red food due was used to identify and exclude the dead spermatozoa from the sorted fractions. The sperm sexing procedure was performed with an SX MoFlo high-speed sorter at a speed of 3000–4000 cells/s. After collecting about 10 million spermatozoa, both fractions, X andY, were mixed, centrifuged at 700g for 15 min to concentrate the spermatozoa (20 million mL–1), and the sperm membranes examined. For sperm membrane examination, 'live/dead' samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20 000 spermatozoa were collected for each sample. The percentage of membrane-intact ('live') spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa stored for 3 days in TALP/BSA v. TALP/Pp was 25.7% (SD = 7.48) v. 28.58% (SD = 7.04), respectively (P < 0.01). The mean percentage of live spermatozoa in samples of sexed semen was 33.57% (SD = 18.97) for TALP/BSA and 38.51% (SD = 20.22) for TALP/Pp (P < 0.01). It can be concluded that Pp should be considered as a replacement for BSA in the TALP medium used for bull sperm sexing because (1) it results in significantly higher numbers of live spermatozoa after storage and/or sexing; (2) it eliminates a possible source of transmissible diseases (such as bovine spongiform encephalopathy); and (3) it decreases the total cost of the basic media used for the bull sperm sexing procedure.
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Sharpe, J. C., and K. M. Evans. "Advances in flow cytometry for sperm sexing." Theriogenology 71, no. 1 (January 2009): 4–10. http://dx.doi.org/10.1016/j.theriogenology.2008.09.021.
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Pindaru, Laura, Iulia Maria Balaci, and Ioan Ştefan Groza. "Sperm sexing technology - new directions in medicine." Revista Romana de Medicina de Laborator 24, no. 1 (March 1, 2016): 111–21. http://dx.doi.org/10.1515/rrlm-2016-0012.
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Seidel, G. E. "Sperm sexing technology—The transition to commercial application." Theriogenology 71, no. 1 (January 2009): 1–3. http://dx.doi.org/10.1016/j.theriogenology.2008.09.015.
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Blecher, S. R., R. Howie, S. Li, J. Detmar, and L. M. Blahut. "A new approach to immunological sexing of sperm." Theriogenology 52, no. 8 (December 1999): 1309–21. http://dx.doi.org/10.1016/s0093-691x(99)00219-8.
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Barros Mothé, Gabriele, Caroline Scott, Carmen Cecília Sicherle, Carlos Renato de Freitas Guaitolini, Camila de Paula Freitas Dell'aqua, Camila Dantas Malossi, João Pessoa Araújo-Júnior, and Fabiana Ferreira de Souza. "Sperm sexing with density gradient centrifugation in dogs." Animal Reproduction Science 199 (December 2018): 84–92. http://dx.doi.org/10.1016/j.anireprosci.2018.11.003.
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Saili, Takdir, La Ode Nafiu, La Ode Baa, Syam Rahadi, Astriana Napirah, Syamsuddin Syamsuddin, I. Wayan Sura, and Febiang Lopulalan. "Efektivitas Sinkronisasi Estrus dan Fertilitas Spermatozoa Hasil Sexing pada Sapi Bali di Sulawesi Tenggara (EFFECTIVENESS OF ESTRUS SYNCHRONIZATION AND SPERMATOZOA FERTILITY RESULTS OF SEXING ON BALI CATTLE IN SOUTHEAST SULAWESI)." Jurnal Veteriner 18, no. 3 (September 4, 2017): 353. http://dx.doi.org/10.19087/jveteriner.2017.18.3.353.
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Estrus synchronization is one of the reproduction technology applied in the cows that aim to induce estrus of some cows to occur in the same time. In this research, all cows expressing estrus would be inseminated using sexed sperm that produced using column albumen method. Sexing sperm technology could be applied to produce the desired sex of calf. Effectivity of chilled sexed sperm to produce the desired sex of calf was evaluated in this research. Sixty three bali cows divided into 2 groups of ages (3-4 yo. and 5- 6 yo.) were used and performed synchronization using Capriglandin (PGF2a) hormone prior to application of artificial insemination with chilled sexed sperm. Variable measured were success rate of synchronization, estrus post synchronization, estrus quality, non return rate, conception rate and calving rate. The results showed that 62.90% of cows showed estrus following synchronization, estrus post synchronization occurred at 71.73 hours following synchronization, and estrus quality was 2.5%. There were 82.54% of inseminated cows was predicted to be pregnant after first insemination using chilled sexed sperm. However, only 73.02% could maintain the pregnancy up to calving. Whereas 78.26 % of newborn calf was male calf. Finally, it was concluded that PGF2a was effective to trigger estrus in bali cows, while sexed sperm still had good fertility and the sex of newborn calf was 78,26% confirmed the prediction.
ABSTRAK
Sinkronisasi estrus merupakan salah satu teknologi reproduksi yang diterapkan pada ternak sapi betina dengan tujuan untuk mendapatkan sejumlah ternak yang estrus secara bersamaan. Pada penelitian ini ternak yang mengalami estrus tersebut diinseminasi menggunakan spermatozoa yang telah melalui proses sexing menggunakan metode kolum albumen. Teknologi sexing spermatozoa memungkinkan untuk mengatur kelahiran anak ternak sesuai jenis kelamin yang diinginkan. Penelitian ini dilakukan untuk mengevaluasi efektivitas penggunaan semen cair hasil sexing dalam memproduksi anak sapi dengan jenis kelamin yang diinginkan. Sapi bali induk sebanyak 63 ekor yang dibagi ke dalam dua kelompok, umur 3-4 tahun dan 5-6 tahun digunakan sebagai akseptor pada penelitian ini. Sebelum inseminasi buatan (IB) dilakukan, semua sapi akseptor disinkronisasi menggunakan hormon Capriglandin (PGF2a). Variabel yang diamati adalah keberhasilan sinkronisasi, estrus pascapenyerentakan birahi, kualitas estrus, non return rate, conception rate dan calving rate. Hasil penelitian menunjukkan bahwa 62,90% sapi mengalami estrus setelah sinkronisasi dengan rataan waktu munculnya estrus 71,73 jam dan kualitas estrus 2,5. Sapi yang diprediksi bunting setelah inseminasi pertama dengan semen hasil sexing mencapai 82,54%. Jumlah sapi yang mampu mempertahankan kebuntingan hingga melahirkan hanya 73,02% dengan persentase jumlah anak sapi jantan yang dilahirkan mencapai 78,26%. Simpulan yang dapat diperoleh dari hasil penelitian ini adalah PGF2a cukup efektif merangsang munculnya estrus pada sapi bali induk dan spermatozoa hasil sexing masih mempunyai daya fertilitas yang cukup baik dengan tingkat kesesuaian jenis kelamin anak sapi yang dilahirkan mencapai 78,26%.
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Amah, Yohanis PIndu, Enike Dwi Kusumawati, and Aju Tjatur Nugroho Krisnaningsih. "The effect of different diluent toward abnormality and motility sexing sperm of etawa cross-bred goat (pe) using egg white sedimentation method." Jurnal Sains Peternakan 5, no. 1 (June 1, 2017): 10–19. http://dx.doi.org/10.21067/jsp.v5i1.3133.
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ABSTRAK
Penelitian ini dilakukan di Balai Besar Inseminasi Buatan Singosari Kabupaten Malang. Tujuan dari penelitian ini adalah untuk mengetahui dan mengkaji pengaruh berbagai metode Sedimentasi putih telur terhadap abnormalitas dan motilitas spermatozoa semen sexing kambing PE. Metode penelitian ini yang digunakan adalah percobaan laboratorium dengan analisis varian. Hasil penelitian menunjukkan pemeriksaan semen berdasarkan evaluasi mikroskopis yang diperoleh menunjukkan gerak massa yang sangat bagus, cepat dan gelap dengan skor positif 3, motilitas individu 94,8%, konsentrasi 3540,2 juta per ml, viabilitas 96,35%, abnormalitas 2,9%. Motilitas spermatozoa dengan menggunakan pengencer Tris lapisan bawah memiliki nilai terendah dibandingkan pengencer lainnya (P<0,01) sebesar 4,71±0,09%. Dapat disimpulkan bahwa abnormalitas dan motilitas spermatozoa semen sexing menggunakan sedimentasi putih telur yang terbaik yaitu dengan pengencer Tris Aminomethan kuning telur dengan rataan motilitas sebesar 65,4% dan rataan abnormalitas sebesar 4,71%.
ABSTRACT
This research was conducted at the Center for Artificial Insemination Singosari district Malang with the purpose of this research is to know and investigate the effect of various methods of white egg sedimentation to abnormalities and motility sexing sperm goat. This research method used a laboratory experiment with analysis was variance. The results showed sperm examination by microscopic evaluation obtained showed a mass movement which was very nice, fast and dark with a positive score of 3, the individual motility was 94.8%, 3540.2 million per ml concentration, viability was 96.35%, abnormalities 2.9 %. Motility was using Tris bottom layer has the lowest value compared to the other diluent (P <0.01) was 4.71 ± 0.09%. It can be concluded that abnormalities and motility sexing sprem using egg whites sedimentation are best used with Tris Aminomethan yolks egg with the average motility was 65.4% and the average abnormalities was 4.71%.
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Rasad, Siti Darodjah, Nurcholidah Solihati, Kikin Winangun, Annisa Yusrina, and Fahmy Avicenna. "Effect of Incubation Time During Sperm Sexing Process on Sperm Quality of Pasundan Bull." Jurnal Ilmu Ternak dan Veteriner 25, no. 3 (September 2, 2020): 112. http://dx.doi.org/10.14334/jitv.v25i3.2494.
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The research was conducted to evaluate the effect of incubation time on viability, plasma membrane integrity, abnormality, and DNA integrity of sexed Pasundan’s bulls sperm. The sperm sexing used 5% and 10% concentrations of Bovine Serum Albumin (BSA). A completely randomized design with three treatments and six replications was used in this study. The data were analyzed using variance analysis followed by Duncan’s multiple distance test. Parameter evaluated were sperm longevity, plasma membrane integrity (PMI), abnormality, and DNA integrity of sexed Pasundan bulls sperm. Results showed that incubation time gave significant effect (P<0.05) on the longevity of sperm, but not on the PMI of Pasundan bulls sexed sperm. The incubation time of 45 minutes gave the highest value of longevity sperm on the upper layer (4.33 days) and the lower layer (4.17 days). Furthermore, the abnormality of sperm X in the upper layer was 4.00%-4.20% and the lower layer was 4.10%- 4.40%. Meanwhile, the DNA integrity of an upper layer was 98.16%-98.66%, and the lower layer was 97.83%-98.58%. It is concluded that 45 minutes of incubation time significantly affected the longevity of sperm, but not plasma membrane integrity, abnormality, and DNA integrity of Pasundan bulls sexed sperm.
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Madrid-Bury, Ninoska, Raúl Fernández, Adela Jiménez, Sonia Pérez-Garnelo, Pedro Nuno Moreira, Belén Pintado, Julio de la Fuente, and Alfonso Gutiérrez-Adán. "Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio." Zygote 11, no. 3 (August 2003): 229–35. http://dx.doi.org/10.1017/s0967199403002272.
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Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.
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Mardi, Irvan, Aulia Puspita Anugra Yekti, Kuswati Kuswati, Muchamad Luthfi, and Trinil Susilawati. "Kualitas Semen Beku Sexing Sapi Peranakan Ongole Menggunakan Volume Semen Awal Yang Berbeda." Jurnal Ilmu dan Teknologi Peternakan Tropis 7, no. 3 (September 25, 2020): 238. http://dx.doi.org/10.33772/jitro.v7i3.12203.
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ABSTRAKInseminasi buatan dengan menggunakan semen sexing diharapkan menghasilkan pedet dengan jenis kelamin sesuai harapan. Tujuan dari penelitian ini adalah untuk mengetahui kualitas, proporsi, dan jumlah produksi straw sexing menggunakan metode sentrifugasi gradien densitas percoll dengan volume awal semen yang berbeda. Penelitian dilakukan di Loka Penelitian Sapi Potong, Kecamatan Grati, Kabupaten Pasuruan dan Laboratorium Reproduksi Ternak, Fakultas Peternakan Universitas Brawijaya, Malang. Materi yang digunakan adalah semen sapi peranakan ongole berumur berkisar lima tahun dan bobot badan berkisar 700 kg sebanyak tiga ekor, motilitas massa ≥2+ dan motilitas individu ≥70%. Metode yang digunakan adalah eksperimental dengan tiga perlakuan volume awal saat sexing, yaitu 1 (P1); 1,5 (P2); dan 2 (P3) ml dengan ulangan 11 kali (ulangan berfungsi sebagai kelompok). Data dianalisa menggunakan Rancangan Acak Kelompok (RAK). Hasil penelitian menunjukkan bahwa perbedaan volume awal semen tidak berpengaruh (menurun) terhadap motilitas, viabilitas, abnormalitas, konsentrasi, total spermatozoa motil, recovery rate dan proporsi spermatozoa (P>0,05). Pengaruh yang sangat nyata (meningkat) terhadap jumlah produksi straw (P<0,01). Ulangan penelitian ini memberikan pengaruh yang sangat nyata (meningkat) terhadap kualitas (motilitas, konsentrasi, viabilitas, abnormalitas, total spermatozoa motil, RR, proporsi dan jumlah straw) dan proporsi spermatozoa X dan Y (P<0,01). Total spermatozoa motil setiap perlakuan telah memenuhi nilai harapan (10 juta/straw). Proporsi spermatozoa X dan Y telah memenuhi nilai harapan (80%:20%).Kata Kunci: kualitas, proporsi, semen beku sexing, strawABSTRACTArtificial insemination using sexing semen is expected to produce calves with the expected sex. The aim of this study was to determine the quality, proportion, and quantity of sexing semen production using the percoll density gradient centrifugation method with different initial semen volumes. The research was conducted at the Beef Cattle Research, Grati District, Pasuruan Regency, East Java Province, Indonesia, and the Animal Reproduction Laboratory, Faculty of Animal Science, University of Brawijaya, Malang, East Java Province, Indonesia. The material used was semen from three Ongole crossbred bull aged around five years and the bodyweight of around 700 kg, mass motility of ≥2+, and individual motility ≥70%. The method used was experimental with three initial volume treatments during sexing, namely 1 (P1); 1.5 (P2), and 2 (P3) ml with 11 replications (replications function as groups). The data were analyzed using a randomized block design (RBD). The results showed that the treatment of differences in initial semen volume did not affect motility, viability, abnormalities, concentration, total motile sperm, recovery rate, and proportion of sperm (P>0.05). On the other hand, the difference in the initial volume of semen had a very significant effect (increased) on the amount of frozen semen production (P<0.01). Repeated research also had a very significant effect (increased) on the semen quality (motility, concentration, viability, abnormality, total sperm motility, recovery rate proportion, and straw production) and the proportion of spermatozoa X and Y (P<0.01). The total motile sperm for each treatment had met the expected value (10 million/straw). Proportions of spermatozoa X and Y have met the expected value (80%: 20%).Keywords: proportion, quality, sexing frozen semen, straw.
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Evans, G., F. K. Hollinshead, and W. M. C. Maxwell. "Preservation and artificial insemination of sexed semen in sheep." Reproduction, Fertility and Development 16, no. 4 (2004): 455. http://dx.doi.org/10.1071/rd04032.
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Sperm-sexing technology using flow cytometry is in advanced stages of development for the sperm of several species. The sorting process could compromise sperm viability and sperm require specific handling procedures both before and after sorting to maintain the integrity and function of the sorted sperm. Standard freezing protocols have been modified for post-sorting cryopreservation of sperm and frozen sperm have been successfully thawed, sorted, refrozen and subsequently used to produce offspring. The relatively low numbers of available sorted sperm have, in some cases, led to modification of artificial insemination techniques to maximise efficiency of use. Multiple ovulation and embryo transfer, or in vitro fertilisation and associated technology, may lead to the more efficient use of sexed sperm.
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Seidel, George E. "Sexing mammalian sperm—intertwining of commerce, technology, and biology." Animal Reproduction Science 79, no. 3-4 (December 2003): 145–56. http://dx.doi.org/10.1016/s0378-4320(03)00162-3.
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Maxwell, W. M. C., G. Evans, F. K. Hollinshead, R. Bathgate, S. P. de Graaf, B. M. Eriksson, L. Gillan, K. M. Morton, and J. K. O’Brien. "Integration of sperm sexing technology into the ART toolbox." Animal Reproduction Science 82-83 (July 2004): 79–95. http://dx.doi.org/10.1016/j.anireprosci.2004.04.013.
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Carvalho, J. O., V. A. Michalczechen-Lacerda, F. C. Rodrigues, R. Sartori, M. M. Franco, and M. A. N. Dode. "278 METHYLATION STATUS IN THE INTRAGENIC DIFFERENTIALLY METHYLATED REGION OF THE IGF2 LOCUS IN UNSORTED AND SEX-SORTED SPERM." Reproduction, Fertility and Development 23, no. 1 (2011): 237. http://dx.doi.org/10.1071/rdv23n1ab278.
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Methylation is the main coordinator of epigenetic inheritance between generations, influencing the regulation of gene expression. Therefore, changes of its pattern in any cell can cause important alterations affecting its function. The methylation pattern of imprinted genes has been reported to be altered by numerous environmental factors such as nutrition, diseases, and drugs. Sexing by flow cytometry exposes the sperm cells to various procedures that can affect sperm quality and could induce methylation changes, resulting in lower fertilization when compared with conventional sperm. Although many studies have related changes in methylation pattern to in vitro culture of oocytes and embryos, no reports have evaluated these changes in sperm caused by the sexing process. The IGF2 imprinted gene, which is predominantly expressed by the paternal allele with the maternal one silenced, is expected to have its intragenic differentially methylated region (DMR) highly methylated in the sperm. Therefore, it became an excellent candidate region to be used for evaluating changes in methylation status of the sperm. The objective of this study was to evaluate the influence of sexing by flow cytometry on the methylation pattern of the DMR located in exon 10 of the IGF2 gene. Frozen–thawed unsorted and sex-sorted sperm samples from 4 Nellore bulls were used (5–10 years old). Each ejaculate was separated into 3 fractions: nonsexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Then, 1 straw of each treatment/bull was thawed, placed in 40:70 Percoll gradient (GE Bioscience®, Uppsala, Sweden) and centrifuged at 700 × g for 45 min to separate the somatic cells from the sperm. Sperm pellets were then used for DNA extraction. After that, DNA was treated with sodium bisulfite using the EZ DNA methylation kit (Zymo Research®, Orange, CA, USA). Bisulfite-treated DNA was amplified in 2-round PCR strategy (nested-PCR) and the amplicons were purified using GenClean III kit (MP Biomedical®, Solon, OH, USA). The purified amplicons were cloned into the pGEM-T easy vector system (Promega®, Madison, WI, USA) and transformed into Escherichia coli cells (XL-1 Blue). The resulting individual clones were sequenced, using a dideoxy fluorescence terminator system (ABI 3130xl, Applied Biosystems, Foster City, CA, USA). The experiment was performed in 3 replicates per bull. Sequences were analysed using the BiQ Analyzer software with a GenBank sequence (X53553) as a reference. At least 60 clones were sequenced per group. The methylation status of the 28 CpG sites was compared among the groups using analysis of variance and Tukey test in the Prophet software. No differences in DNA methylation were found between NS (97.5 ± 1.7%), SX (95.8 ± 1.8%), and SY (97.3 ± 0.2%) (P = 0.36). Moreover, 100% of analysed sequences in all groups were hypermethylated. Therefore, we can conclude that the process of sexing by flow cytometry does not change the methylation pattern in the intragenic DMR located in exon 10 of the IGF2 gene. Financial support: FAPESP and Embrapa.
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Kątska-Książkiewicz, L., B. Ryńska, M. Bochenek, J. Opiela, and J. Jurkiewicz. "In vitro production of bovine embryos using flow-cytometrically sexed sperm." Archives Animal Breeding 49, no. 2 (October 10, 2006): 133–40. http://dx.doi.org/10.5194/aab-49-133-2006.
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Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.
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Knijn, H. M., A. C. J. Frijters, J. E. Roelfzema, M. Bakker, R. Lenz, and J. S. Merton. "369 EFFECT OF HOLDING TIME OF RAW HOLSTEIN FRIESIAN BULL EJACULATES BEFORE SEXING ON NON-RETURN RATES AFTER 56 DAYS." Reproduction, Fertility and Development 22, no. 1 (2010): 341. http://dx.doi.org/10.1071/rdv22n1ab369.
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Flow cytometric seperation of X- and Y-chromosme-bearing sperm is the only reliable technique that has been used succesfully for commercialization of sexed semen in the dairy industry. However, sperm sexing results in lower fertility compared to conventional semen. Due to logistics like the distance between bull station and sorting laboratory, the number of flow cytometers, and the time needed for the sorting process itself, the holding time between ejaculation and freezing of sex-sorted semen is variable. This holding time could influence the fertility of sex-sorted semen. The aim of this study was to evaluate the effect of different holding times before sexing on the non-return rates on Day 56 post insemination (NRR56). Following collection, raw ejaculates were transported at room temperature (19°C) from two semen collection centers to the central sorting laboratory. Within 2-4 h after collection, volume, sperm concentration and quality of the ejaculates were determined. When accepted, aliquots of 1 mL of semen were stained every 45-60 min for 2 h. Subsequently, sexing was performed according to procedures described previously, using MoFlo SX™ sperm sorters (Garner DL and Seidel GE 2008 Theriogenology 69 : 886-895). Each aliquot of 1 mL semen was sorted for 45-60 min. Four flow sorters were used, so not more than 4 aliquots could be processed simultaneously. After sorting 3-4 h, semen was pooled and a charge number was assigned to this fraction of the ejaculate (called a freezerun). This procedure was done a maximum of 3 times. The time between collection of the raw ejaculate and freezing of the semen was 7-10 h for freezerun 1, 10-14 h for freezerun 2, and 13-18 h for freezerun 3. From September 2007 to April 2009, a total of 16,523 inseminations with sexed semen from 43 different Holstein bulls were performed for which non-return rates at 56 days (NRR 56) were recorded. There were no significant differences in NRR56 between the 3 freezeruns (t-test) (Table 1). The results shown in this study suggest that raw Holstein semen can be held at room temperature for up to 13-18 h prior to sexing and freezing without an effect on NRR56. This study did not indicate what the maximum holding time prior to sexing and freezing would be before resulting in a fertility decline. Table 1.Number of records and NRR56 for different freezeruns
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Morton, K. M., S. L. Catt, F. K. Hollinshead, W. M. C. Maxwell, and G. Evans. "280LAMBS BORN AFTER IN VITRO EMBRYO PRODUCTION FROM PREPUBERTAL LAMB OOCYTES AND FROZEN-THAWED UNSORTED AND SEX-SORTED SPERMATOZOA." Reproduction, Fertility and Development 16, no. 2 (2004): 260. http://dx.doi.org/10.1071/rdv16n1ab280.
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Developments in sperm sexing technology have resulted in the birth of a number of offspring after IVF of oocytes from adult animals (Johnson LA, 2000 An. Reprod. Sci. 60–61, 93–107). The aim of this study was to combine sperm sexing technology with juvenile breeding. Merino lambs, 2–3 weeks (n=43) were hormone stimulated (Morton KM et al., 2003 Proc. Soc. Reprod. Fert., P18), and COCs were matured in TCM-199 (Sigma) with 10μgmL−1 p-FSH (Folltropin-V; Bioniche Animal Health Australasia), 10μgmL−1 pLH (Bioniche), and 20% sheep serum (v/v) in a humidified 6% CO2, 5% O2, 89% N2 atmosphere for 22h. Semen collected from Merino rams was diluted and frozen as pellets (Unsorted), or stained with H33342, separated into X and Y sperm using a SX MoFlo (Cytomation Inc., Fort Collins, CO, USA), and frozen as pellets (Sorted). Sperm were prepared for IVF by swim-up under 0.5mL of SOF with 2% sheep serum (v/v; SOF+) for 45min (Unsorted), or diluted in 0.5mL of Sydney IVF Sperm Buffer (Cook IVF, Brisbane, Australia) and centrifuged at 650g for 3min (Sorted). After IVM, oocytes were transferred to SOF+, and cultured with 0.5×106mL−1 (Unsorted) or 1.0×106mL−1 (Sorted) motile sperm for 18h. Presumptive zygotes were transferred to Sydney IVF cleavage and blastocyst medium (Cook IVF) for 3 and 5 days, respectively. Oocyte maturation and fertilization were assessed by orcein staining 18h post-insemination (hpi). Two Day-7 blastocysts were transferred to each recipient ewe (n=9; 3 per group) and pregnancies diagnosed by ultrasound on Day 57 of gestation. Data were analyzed by chi-square test. Oocyte maturation was 83.9% (73/87), and monospermic fertilization did not differ for Unsorted (22/32; 68.7%), X- (6/14; 42.8%), and Y-sperm groups (15/27; 55.6%). Polyspermic fertilization was 9.4% (3/32) and 7.4% (2/27) for the Unsorted and Y groups. Cleavage was reduced with X- and Y-sperm compared with Unsorted, but blastocyst formation (from cleaved oocytes) did not differ (Table 1). There were three (100%), zero (0%), and one (33.3%) pregnancies from Unsorted, X- and Y-embryos, respectively, all of which survived to birth, demonstrating that juvenile breeding can be successfully combined with sperm sexing. Table 1 Cleavage and blastocyst formation after IVF with Unsorted, X- or Y-sperm. Values in parenthesis are percentages
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Avelino, K. B., M. R. Rossetto, A. C. Maraia, M. R. Lima, A. T. R. Mansano, and J. M. Garcia. "262 REPORTS OF IN VITRO PRODUCTION AND PREGNANCY RATES OF BOVINE EMBRYOS PRODUCED BY POST-THAWING SEXED SEMEN." Reproduction, Fertility and Development 22, no. 1 (2010): 288. http://dx.doi.org/10.1071/rdv22n1ab262.
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Births of predetermined-sex calves illustrates the importance of sperm sexing technology for in vitro production of bovine embryos (Johnson, 2000). Flow cytometry techniques for sperm sexing are usually performed on fresh semen. However, many bulls presenting high genetic value died or became unproductive before development of sperm sexing technologies. The technique of fresh semen sexing and semen reverse sorting method is based on separation of sperm according to differences in DNA content (X or Y). The objective of this study is to examine the blastocysts and pregnancy rates of bovine embryos produced by fertilization with sperm sexed with the post-thawing procedure, called reverse sorting. Oocytes were obtained by ovum pick-up from Dairy Gir, Nelore, and Guzera cows. The COC were transported to the laboratory and matured at 38.5°C and 5% of CO2 in air, TCM-199 medium with FCS (10% vol/vol), FSH (1.0 μg mL-1), hCG (50 μg mL-1), estradiol (1.0 μg mL-1), sodium pyruvate (0.20 mM), and amicacin (83.4 μg mL-1). IVF was performed 24 to 26 h after the onset of maturation. Doses of commercialized semen (3 to 5) from the specified breeds were sent to Goyaike Brazil Agropecuária Ltda. The semen sexing process was performed by flow cytometry and semen was returned to the IVF laboratory at 18°C. In the Tecgene laboratory, semen was centrifugated using Percoll gradient and evaluated. After 18 h, the presumptive zygotes were transferred to IVC in SOF medium at 38.5°C and 5% CO2 in air. The cleavage rate was evaluated 48 h after IVF and embryos were transferred at Day 7. A total of 5213 viable oocytes were obtained from 266 donors, fertilized with 7 different reverse sorted semen samples (1 Dairy Gir, 2 Nelore, and 4 Guzera) resulting in 1333 embryos. From these, 1084 transferred embryos resulted in 260 pregnancies to this time, with 226 females and 34 males. Each bull was evaluated separately according to cleavage rate, production of blastocysts, pregnancy rate, and percentage of females, respectively. Bull 1 presented 100%, 38%, 25%, and 90%; bull 2: 98%, 38%, 21%, and 90%; bull 3: 84%, 23%, 21%, and 84%; bull 4: 87%, 15%, 29%, and 86%; bull 5: 92%, 53%, 70%, and 71%; bull 6: 78%, 15%, 34%, and 71%, and bull 7: 55%, 27%, 50%, and 78%. The average of in vitro blastocysts (26%), the pregnancy rate (24%), and the percentage of females (87%) are similar to rates obtained by commercial fresh semen sexed by Goyaike Brazil Agropecuária Ltda. In conclusion, semen reverse sorting is an alternative method for selecting sex of in vitro production of bovine embryos using thawed semen collected from extinct or unproductive bulls.
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Ashari, Lukman, Imam Mustofa, Maya Nurwartanti Yunita, Trilas Sardjito, Amung Logam Saputro, and Ragil Angga Prastiya. "Pengaruh Durasi Waktu Pada Sexing Spermatozoa Sapi Bali Terhadap Kualitas Dan Efektivitas Sexing Spermatozoa Dengan Menggunakan Alat Electric Separating Sperm (ESS)." Jurnal Medik Veteriner 2, no. 1 (March 31, 2019): 24. http://dx.doi.org/10.20473/jmv.vol2.iss1.2019.24-29.
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Tujuan penelitian ini adalah mengetahui perbedaan kualitas (viabilitas, motilitas dan abnormalitas) spermatozoa Sapi jantan Bali hasil sexing menggunakan alat ESS yang dialiri listrik selama lima menit dan sepuluh menit pada sisi anoda dan katoda dan mengetahui perbedaan efektivitas pemisahan spermatozoa Sapi Bali hasil sexing menggunakan alat ESS yang dialiri listrik selama lima menit dan sepuluh menit pada sisi anoda dan katoda. Penelitian ini menggunakan Sapi Bali yang berumur 4 tahun sampai 7 tahun dengan motilitas di atas 45% dan di bawah 60%. Penelitian ini menggunakan diluter tris kuning telur. Sexing spermatozoa menggunakan alat ESS yang dialiri listrik dengan durasi 5 menit dan 10 menit pada masing-masing anoda dan katoda. Hasil penelitian ini menunjukan kualitas (viabilitas, motilitas dan abnormalitas) mengalami penurunan. Kualitas terbaik pada sexing spermatozoa terdapat pada durasi lima menit. Efektivitas pemisahan spermatozoa yang paling baik yaitu pada durasi sepuluh menit pada sisi katoda spermatozoa X sebesar 62,17±0,240% dan pada sisi anoda spermatozoa Y sebesar 67,33± 1,03%.
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Suzuki, Hiroshi, and Maya Oi. "Sperm Sexing in the Dog by Fluorescence In Situ Hybridization." Biology of Reproduction 87, Suppl_1 (August 1, 2012): 556. http://dx.doi.org/10.1093/biolreprod/87.s1.556.
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Bochenek, M., T. Herjan, and Z. Smorag. "361 INFLUENCE OF SEXING PROCEDURE ON BULL SPERM CHROMATIN STRUCTURE." Reproduction, Fertility and Development 19, no. 1 (2007): 296. http://dx.doi.org/10.1071/rdv19n1ab361.
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Flow cytometry is the only reliable and relatively fast method allowing separation of live X and Y spermatozoa for sex regulation. Many thousands of animals of different mammalian species have been born after insemination with sexed semen during the past 20 years. Nevertheless, the question is still open: does the bull sperm sexing technology affect chromatin structure? A case of serious chromatin damage after sexing stallion semen was reported previously (Bochenek et al. 2006 Havemeyer Foundation Monograph Series No. 18, 13 –14). The aim of this work was to examine the effect of the sexing procedure and different UV laser powers on bull sperm chromatin structure. The ejaculates of 28 bulls (one ejaculate/bull) were used in the study. Each ejaculate was divided into 5 groups: (1) control, unprocessed; (2) sorted strictly according to XY Inc. protocol (Schenk et al. 1999 Theriogenology 52, 1375 –1391); (3) as group 2, but without the Red Food dye staining used for dead spermatozoa discrimination; (4) as group 2, but with double UV laser power (300 mW); and (5) as group 3, but with double UV laser power (300 mW). Sperm sorting was performed with a MoFLoSX flow cytometer at speeds of 3000 –5000 cells/s. Sorted fractions of X and Y spermatozoa were mixed again and stored for 24 h at 15 °C. A sperm chromatin structure assay (SCSA) was performed twice on each sorted sample, immediately after sorting and after 24 h. The chromatin of control samples was examined according to the same time schedule. The percentage of spermatozoa with damaged chromatin was calculated (COMP α-t) as well as standard deviation of the α-t parameter (SD α-t). The latter parameter, although less intuitive, is considered as even more precise than COMP α-t in chromatin investigations. The mean percentage of spermatozoa with abnormal chromatin was 1.12% (SD = 0.47) for control samples. The highest level of chromatin abnormality was noted for the 300 mW group with no dead cell discrimination (Red Food staining): 1.29% (SD = 1.05). After 24 h of storage, the mean level of chromatin abnormality increased to 1.97% (SD = 0.96) in control samples whereas that in all sorted samples was lower: from 1.06% (SD = 0.4) to 1.16% (SD = 0.62) in the 150 mW/non-Red Food-stained and the 300 mW/Red Food-stained groups, respectively. This difference appeared to be statistically significant (t; P ≤ 0.05). Interestingly, the percentage of abnormal spermatozoa decreased slightly after 24 h of storage in the 300 mW/Red Food-stained and the 300 mW/non-Red Food-stained groups ( –0.13% and –0.08%, respectively). Calculation of the SD α-t parameter showed statistically significant differences in chromatin abnormality between the control group vs. the 300 mW/non-Red Food-stained group immediately after sorting and the control group vs. the 150 mW/Red Food-stained group after 24 h of storage. In conclusion, although the statistically significant increase of chromatin damage was found after sexing in some investigated groups, it seems that the level of this abnormality is far too low to affect sexed semen fertility.
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Rai, Jagdish. "Sperm Sexing of Dairy Cattle:Economics, Animal Welfare and Technological Challenges." Current Science 114, no. 07 (April 10, 2018): 1438. http://dx.doi.org/10.18520/cs/v114/i07/1438-1442.
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SEIDEL, Jr., George E. "Sexing Mammalian Sperm ^|^ndash; Where Do We Go from Here?" Journal of Reproduction and Development 58, no. 5 (2012): 505–9. http://dx.doi.org/10.1262/jrd.2012-077.
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Tapaloaga, D., and P. R. Tapaloaga. "Challenges and upcoming developments in sperm sexing for cattle industry." Journal of Biotechnology 305 (November 2019): S10. http://dx.doi.org/10.1016/j.jbiotec.2019.05.051.
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González-Marín, C., A. L. Travis, M. E. Kjelland, J. Gosálvez, C. López-Fernández, R. W. Lenz, and J. F. Moreno. "280 BOVINE SPERM DNA FRAGMENTATION RATES AND EXTENDER pH: A DYNAMIC EXPERIMENTAL APPROACH." Reproduction, Fertility and Development 23, no. 1 (2011): 238. http://dx.doi.org/10.1071/rdv23n1ab280.
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Flow cytometry technology for the sorting of X- and Y-chromosome bearing sperm is currently utilised in research and for commercial applications. During sample preparation before the sex-sorting process, sperm go through different pH treatments that may affect their quality. The hypothesis to test in this study is that sperm DNA damage could occur due to differences in the pH of sperm extenders. Bull semen doses from 15 dairy and 15 beef bulls (2 ejaculates per individual), ranging in age between 13 and 96 months, were randomly selected from a bull stud in Texas (Sexing Technologies, Navasota, TX, USA). Each semen sample was divided into 4 separate aliquots. One neat semen sample was kept as a control and the other 3 aliquots were treated with 16 μL of 8.1 mM Hoechst 33342 (Molecular Probes, Eugene, OR, USA). A calculated amount of modified Tyrode’s albumin lactate pyruvate (Clear TALP), pH 7.4, was added based on neat ejaculate concentration. For the separation of live and dead sperm during the sex-sorting process, 2 mL of red TALP (Red Food Dye FD&C #40; Sensient Technologies Corp., Milwaukee, WI, USA) at 3 different pH treatment levels (5.5, 6.4, and 7.4) were added to the sperm samples. The dynamics of sperm DNA fragmentation were assessed immediately following the addition of red TALP treatment and after 24 h of incubation at 34°C. Sperm DNA fragmentation was measured using the commercial variant of the sperm chromatin dispersion test, the bull Sperm-Halomax® kit (Halotech DNA, Madrid, Spain), and counting 300 sperm cells under fluorescence microscopy. Analysis of variance was used to determine if there were statistical differences (α = 0.05) among mean values of the groups (SPSS v.17.0 for Windows, SPSS Inc., Chicago, IL, USA). Bonferroni post hoc tests were utilised to determine the pairwise directional differences between groups. Differences in DNA fragmentation were not observed at 0 h (P > 0.05); however, at 24 h this value was significantly higher (P < 0.05) in semen samples treated with red TALP, pH 6.4, v. samples treated with red TALP, pH 7.4, (44.0 ± 24.9% v. 34.8 ± 24.9%), and even higher when using red TALP pH 5.5 (50.8 ± 23.8%). Further experiments should be designed to demonstrate that the DNA molecule could be affected by pH fluctuations, which may be important in refining the sex-sorting process. Notably, the undiluted neat semen controls had greater levels of DNA fragmentation overall (57.5 ± 29.4%), suggesting a negative effect of high sperm concentrations and seminal plasma. The authors thank Mike Evans, Eddy Valenzuela, David Del Olmo, Jamie Jeffers, and the staff at Sexing Technologies for their technical assistance. This research was funded by Sexing Technologies and XY Inc.
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O'Brien, J. K., F. K. Hollinshead, G. Evans, and W. M. C. Maxwell. "332IN VIVO DEVELOPMENTAL CAPACITY OF IN VITRO-PRODUCED EMBRYOS DERIVED FROM SEX-SORTED AND RE-CRYOPRESERVED FROZEN-THAWED RAM SPERM." Reproduction, Fertility and Development 16, no. 2 (2004): 286. http://dx.doi.org/10.1071/rdv16n1ab332.
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The ability to sort and re-freeze frozen-thawed sperm would significantly increase the potential application of sperm sexing technology to species management. Frozen-thawed, sorted, re-frozen and then thawed ram sperm appear fully functional in vitro with blastocyst production greater than that for frozen-thawed, non-sorted sperm (Hollinshead FK et al. 2003 Theriogenology 59, 209 abst). The aim of this study was to evaluate the in vivo capacity of in vitro-produced embryos derived from frozen-thawed sperm after sorting and a second cryopreservation/thawing step. Frozen semen from 2 rams (n=3 ejaculates per ram) was used throughout. Post-thaw sperm treatments comprised (i) non-sorted (Control); (ii) sorted (Froz-Sort) and (iii) sorted, then re-frozen (Froz-Sort-Froz). X and Y sperm were separated using a high-speed sorter (SX MoFlo®, DakoCytomation, Fort Collins, CO, USA) after incubation with Hoechst 33342 and food dye to eliminate nonviable sperm. Reanalysis revealed high levels (mean±SEM) of purity for X- and Y-enriched samples for all treatments (89±1.2%). At Day 6 post-insemination, 2 embryos (blastocyst stage or greater) were transferred per recipient. Data were analyzed by chi-square and Fisher Exact Test. In vivo embryo survival was similar across sperm treatments (28/64, 43.8% overall) and 20 of 23 (87.0%) sexed lambs were of the predicted sex (Table 1). These results demonstrate high in vivo developmental capacity of in vitro-produced sexed embryos derived from frozen-thawed ram sperm after sorting and a second cryopreservation/thawing step, and increase the potential application of sperm sexing technology. Research support by XY, Inc., Australian Research Council and Zoological Parks Board of NSW. Table 1 In vivo survival of transferred in vitro produced embryos derived from frozen-thawed non-sorted (Control), frozen-thawed and sorted (Froz-Sort) and frozen-thawed, sorted, then frozen-thawed (Froz-Sort-Froz) ram sperm.
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Schenk, J. L., Z. Brink, and T. K. Suh. "310 USE OF COMPETITIVE FERTILIZATION TO EVALUATE A SIMPLER LASER FOR FLOW CYTOMETRIC SEXING OF BOVINE SPERM." Reproduction, Fertility and Development 17, no. 2 (2005): 306. http://dx.doi.org/10.1071/rdv17n2ab310.
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Statistically significant correlations between laboratory assays of sperm quality and fertility require large sample populations. Experimental differences can be assessed accurately using limited observations with competitive fertilization and fetal sexing, as clearly demonstrated previously (Seidel GE Jr. et al. 2003 Theriogenology 59, 515 abst). Our objectives were to compare pregnancy rates in Holstein heifers inseminated with: (1) 2 × 106 hetero-sex-selected sperm interrogated with different light sources or, (2) 2 × 106 or 10 × 106 X-chromosome bearing sperm using a continuous wave (CW) laser, to those of unsexed inseminates containing 10 × 106 total sperm. Sperm were sexed by flow cytometry/cell sorting at 40 psi with the aid of light produced from either a continuous wave (CW) or a quasi-cw (PULSED; Vanguard 350-HDM, Spectra-Physics, Mountain View, CA, USA) laser operating at 150 mW during flow cytometric/cell sorting. Subsequent fertility was evaluated by competitive fertilization with fetal sex as the genetic marker. Sperm from Holstein bulls were sorted into X- and Y-chromosome populations at >90% accuracy using either the CW or the PULSED laser. After concentration of sperm post-sorting by centrifugation, an equal number of X-sorted sperm illuminated with the CW laser were pooled with Y-sorted sperm illuminated by the PULSED laser within each bull, as well as the converse. Total sorted sperm (2 × 106) were placed in 0.25 mL straws. In addition to these sperm, homospermic inseminates containing 2 or 10 × 106 total sperm, sorted using the CW laser, and unsorted controls (10 × 106 total sperm) were then frozen. Holstein heifers (n = 763) were synchronized for estrus in five groups (July–December) with a CIDR in place for 7 days followed by 25 mg PGF-2α i.m. Heifers (n = 626) were body inseminated either 12 or 24 h after observed estrus. Sexed and unsorted inseminates were balanced across sperm from three bulls and two inseminators. Two months post-insemination, pregnancy and fetal sex were determined using ultrasound. Data were subjected to ANOVA. Pregnancies marked by fetal sex achieved with competitive fertilization resulted in a 52 (PULSED):48 (CW) ratio, which is not different from the expected 50:50 ratio (P > 0.05) if neither laser was more or less damaging to the fertilizing potential of sperm. Actual pregnancy rates for the competitive 2 × 106, homospermic 2 × 106, and 10 × 106 sexed inseminates were 54, 56, and 62%, respectively, with n = 179, 179, and 180, and were similar to unsorted controls (61%, n = 88) (P > 0.05). This study demonstrated no deleterious effects, in terms of pregnancy rate, to sperm illuminated with a PULSED laser during sorting when compared to conventional instrumentation. Also, pregnancy rates with sexed sperm were similar to those of unsexed controls. The use of the PULSED laser for sperm sorting has economic advantages because it requires less energy, is air-cooled, and has a longer operating life than the water cooled CW laser.
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Anema, J. L., J. K. Graham, R. W. Lenz, and G. E. Seidel. "362 STORAGE OF BOVINE SPERM FOR 20 H BETWEEN SEMEN COLLECTION AND SEXING." Reproduction, Fertility and Development 22, no. 1 (2010): 337. http://dx.doi.org/10.1071/rdv22n1ab362.
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The objective of this study was to optimize bovine sperm storage for up to 20 h between semen collection and sex sorting followedby cryopreservation. Two successive ejaculates were obtained from mature dairy bulls (Holstein, n = 5; Jersey, n = 3) via artificial vagina. Treatments were then applied to the neat semen to which antibiotics were added as recommended by Certified Semen Services (Columbia, MO). Nothing further was added to the control samples until staining with Hoechst 33342 for sorting. For Treatment 1, semen was diluted 9:1 with a MOPS solution resulting in 24 mM MOPS and similarly, Treatment 2 resulted in 24 mM MOPS +2% egg yolk. A subsample of each treatment and control was sorted by flow cytometry shortly after collection, and sperm then were frozen following standard processing procedures. The other subsample was stored at 15-18°C and sorted 20 h after collection followed by cryopreservation. pH measurements were made before staining samples for sorting. Samples were evaluated post-thaw for subjective progressive and total sperm motility, by computer-assisted sperm analysis (CASA, Berkeley, CA, USA), and by flow cytometry for sperm viability using propidium iodide and SYBR-14. Treatment 1 performed better than the control (Table 1), while results for Treatment 2 were similar to the control. Second ejaculates were superior to first ejaculates. pH measurements showed that addition of MOPS kept the pH about 0.2 units higher than the control, but pH declined similarly over time in all groups. While responses for the 20 h sort were numerically lower than the 0 h sort (P > 0.1), the majority of responses were acceptable for most, but not all bulls. In conclusion, storing sperm in 24 mM MOPS was beneficial. Surprisingly, 2% egg yolk negated the beneficial effect of MOPS, possibly due to increasing osmolarity by ∼15mOsM/kg due to pH adjustment. Addition of MOPS provided better results than the control for both the 0 h and 20 h sorts. Table 1.Main effect means of semen characteristics
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Gonzalez-Marin, C., R. W. Lenz, T. B. Gilligan, K. M. Evans, C. E. Gongora, J. F. Moreno, and R. Vishwanath. "191 SexedULTRA™, A NEW METHOD OF PROCESSING SEX SORTED BOVINE SPERM IMPROVES POST-THAW SPERM QUALITY AND IN VITRO FERTILITY." Reproduction, Fertility and Development 29, no. 1 (2017): 204. http://dx.doi.org/10.1071/rdv29n1ab191.
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Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P < 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm. Table 1. Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method
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Katska-Ksiazkiewicz, L., M. Bochenek, B. Rynska, and J. Opiela. "307 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING FLOW-CYTOMETRICALLY SORTED SPERMATOZOA." Reproduction, Fertility and Development 17, no. 2 (2005): 304. http://dx.doi.org/10.1071/rdv17n2ab307.
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There are two practical ways to predetermine the sex of mammalian offspring: sexing pre-implantation embryos and sexing spermatozoa. The only successful and non-invasive method of sexing spermatozoa is quantifying sperm DNA with fluorescing DNA-binding dye, followed by flow cytometry and cell sorting. Our investigations aimed to develop a technology for in vitro embryo production in cattle using fresh and/or frozen-thawed spermatozoa sexed by flow cytometry. Sperm was sorted in a MoFloSX® cytometer using the method of XY, Inc. (Fort Collins, CO, USA; Research Collaboration Agreement). After sorting, the sperm was either used for IVF or frozen and stored in liquid nitrogen. Immature oocytes, recovered from slaughterhouse ovaries, after 22 to 23 h of IVM in TCM-199 containing 20% estrous cow serum and additional granulosa cells (Katska et al. 1998, J. Anim. Feed Sci. 7, 353–362), were fertilized in vitro with fresh or frozen-thawed X and Y fractions of spermatozoa. Simultaneously control, unsorted, fresh and frozen-thawed sperm was used for IVF. The standard protocol of sperm capacitation (Katska and Rynska 1998 Theriogenology 50, 213–222) was applied for both control sperm and fresh fractions of sexed sperm. Briefly, sperm was separated by Percoll gradient centrifugation, washed, and introduced into drops of Tyrode's albumin-lactate-pyruvate (TALP)-IVF (containing 10 μg heparin mL−1 and mixture of penicillamine, hypotaurine, and epinephrine) at a concentration 1 to 2 × 106 spermatozoa mL−1 of medium. Frozen fractions of sorted spermatozoa were centrifuged after thawing (500 g for 10 min) and immediately introduced into the IVF drops at 2 to 3 × 106 spermatozoa mL−1 of medium. Embryos resulting after IVF were co-cultured with Vero cells in B2 medium supplemented with 2.5% fetal calf serum for 8 to 10 days, (i.e., to the hatched blastocyst stage). A total of 2074 IVM oocytes were fertilized with both fresh and frozen-thawed sexed and control sperm of 5 bulls. There were significant differences (P < 0.01) in cleavage rates among fresh control sperm (120/256; 46.9%), the X fraction (66/254; 26.0%), and the Y fraction (58/230; 25.2%). Similar differences in cleavage rates (P < 0.01) were shown for frozen-thawed control sperm (156/335; 46.6%), the X fraction (137/498; 27.5%), and the Y fraction (118/501; 23.6%). No differences were observed in efficiency of embryo development to the blastocyst stage between the fresh control (25.8%) and the Y fraction (25.9%), or among the frozen control (16.7%) and the X fraction (13.1%) or the Y fraction (16.9%). However, significant differences (P < 0.05) were shown between blastocyst rates with the fresh X fraction (10.6%) and the control. Our results suggest that there were differences due to sperm sorting but no differences in efficiency of both fresh and frozen-thawed X and Y fractions of spermatozoa. Research was supported by the State Committee for Scientific Research as a project 3PO6D 044 23.
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Johnson, Lawrence A., Glenn R. Welch, and Wim Rens. "The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI." Journal of Animal Science 77, suppl_2 (1999): 213. http://dx.doi.org/10.2527/1999.77suppl_2213x.
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Didion, B. A., and R. Bleher. "261 LABELING SEX-SPECIFIC DNA SEQUENCES IN MAMMALIAN SPERM." Reproduction, Fertility and Development 20, no. 1 (2008): 210. http://dx.doi.org/10.1071/rdv20n1ab261.
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While flow cytometric separation of X- andY-chromosome- bearing sperm has advanced to the point of acceptance in the commercial production of sex-preselected cattle, it is important to continue researching this area to improve efficiencies. For example, the difference in DNA sequence between the X- andY-chromosomes has merit as a foundation for an alternative sperm sexing approach that could enable the complete separation and use of an entire ejaculate. We used synthetic DNA mimics conjugated to a fluorescent dye for in situ detection of Y-chromosomes in metaphase preparations of porcine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are synthetic compounds with higher affinity and stability than conventional DNA probes and are used as specific hybridization probes to complementary DNA. The application of PNA probes was demonstrated previously in telomere analysis studies, and we confirmed their efficacy using a CY3-(CCCTAA)3 PNA to probe bull and boar sperm telomeric sequences. Using male porcine somatic cells and theY-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13-15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on theY-chromosome. No signals were present when chromosomes of porcine female somatic cells were incubated with the PNA probes. Because chromosomes occupy non-random territories in all cell nuclei including those in sperm, we expected to find centrally located signals in 50% of fixed boar sperm when these were treated with the same PNA as used for the somatic cells. We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally located, round fluorescent dot in the sperm head. Further research is required to establish the uptake of PNA in live sperm toward evaluation of this approach for sperm sexing.
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de Graaf, S. P., K. H. Beilby, S. L. Underwood, G. Evans, and W. M. C. Maxwell. "Sperm sexing in sheep and cattle: The exception and the rule." Theriogenology 71, no. 1 (January 2009): 89–97. http://dx.doi.org/10.1016/j.theriogenology.2008.09.014.
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Vanthournout, B., K. Deswarte, H. Hammad, T. Bilde, B. Lambrecht, and F. Hendrickx. "Flow cytometric sexing of spider sperm reveals an equal sperm production ratio in a female-biased species." Biology Letters 10, no. 5 (May 2014): 20140159. http://dx.doi.org/10.1098/rsbl.2014.0159.
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Producing equal amounts of male and female offspring has long been considered an evolutionarily stable strategy. Nevertheless, exceptions to this general rule (i.e. male and female biases) are documented in many taxa, making sex allocation an important domain in current evolutionary biology research. Pinpointing the underlying mechanism of sex ratio bias is challenging owing to the multitude of potential sex ratio-biasing factors. In the dwarf spider, Oedothorax gibbosus , infection with the bacterial endosymbiont Wolbachia results in a female bias. However, pedigree analysis reveals that other factors influence sex ratio variation. In this paper, we investigate whether this additional variation can be explained by the unequal production of male- and female-determining sperm cells during sperm production. Using flow cytometry, we show that males produce equal amounts of male- and female-determining sperm cells; thus bias in sperm production does not contribute to the sex ratio bias observed in this species. This demonstrates that other factors such as parental genes suppressing endosymbiont effects and cryptic female choice might play a role in sex allocation in this species.
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Ama, Kornelis Tamo, Enike Dwi Kusumawati, and Aju Tjatur Nugroho Krisnaningsih. "Kualitas spermatozoa semen sexing kambing peranakan etawa (pe) dengan metode sedimentasi putih telur menggunakan pengencer yang berbeda." Jurnal Sains Peternakan 5, no. 1 (June 1, 2017): 39–49. http://dx.doi.org/10.21067/jsp.v5i1.3136.
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ABSTRAK
Tujuan penelitian ini adalah untuk mengetahui kualitas spermatozoa semen hasil sexing kambing Peranakan Etawa (PE) dengan metode sedimentasi putih telur menggunakan pengencer yang berbeda. Materi yang digunakan dalam penelitian ini adalah semen segar kambing PE. Metode percobaan yang digunakan adalah percobaan laboratorium dengan menggunakan rancangan acak lengkap (RAL) Setiap perlakuan semen sexing kambing Peranakan Etawa dengan pengencer yang berbeda yaitu (P0) CEP, (P1) CEP+kuning telur+putih telur, (P2)CEP+putih telur (P3), CEP+kuning telur dan diulang sebanyak 10 kali. Variabel yang diukur pada penelitian ini adalah motilitas, viabilitas, dan abnormalitas spermatozoa. Data yang diperoleh dianalisis dengan mengunakan analisis varian. Apabila perlakuan memberikan pengaruh maka dilanjutkan dengan uji Beda Nyata Terkecil (BNT). Hasil penelitian menunjukkan bahwa kualitas spermatozoa kambing Peranakan Etawa (PE) dengan berbagai macam pengencer menunjukkan pengaruh yang sangat nyata (P<0,01). Motilitas dan Viabilitas menunjukkan perbedaan yang sangat nyata (P<0,01) pada berbagai pengencer Motilitas terbaik pada beberapa lapisan atas sebesar 65% dan CEP+PT lapisan atas 60% dilanjutkan oleh CEP+KT+PT lapisan bawah 55,9% dan CEP+PT lapisan bawah 55% dilanjutkan lagi oleh CEP+KT lapisan atas 50% dan lapisan bawah 45% dan yang paling terkecil adalah CEP lapisan bawah 40%. Viabilitas spermatozoa dari yang tertinggi yaitu CEP+KT+PT lapisan atas sebesar 69,553% dan CEP+PT lapisan atas sebesar 69,519% dan dilanjutkan dengan CEP+PT lapisan bawah sebesar 65,504% dan CEP+KT+PT lapisan bawah 65,473%, dan dilanjutkan lagi dengan CEP+KT lapisan atas sebesar 60,269% dan lapisan bawah sebesar 53,476% dan yang paling terkecil yaitu CEP lapisan bawah sebesar 40,371%. Presentase Abnormalitas menunjukkan bahwa perlakuan tidak memberikan pengaruh nyata (P>0,05). Tetapi CEP+KT+PT lapisan atas menunjukkan persentase yang paling rendah yaitu sebesar 4,88% dibandingkan perlakuan lainnya. Dari hasil penelitian dapat disimpulkan bahwa kualitas spermatozoa semen sexing kambing PE dengan menggunakan pengencer CEP+ kuning telur + putih telur pada lapisan atas memberikan hasil terbaik terhadap kualitas spermatozoa ditinjau dari motilitas, viabilitas dan abnormalitas.
ABSTRACT
The purpose of this study was to determine the sexing sperm quality of Etawa cross-bred goat (PE) with egg white sedimentation method using different diluents. The material used in this study was the fresh semen Etawa crossbreed goat (PE) of the center for Artificial Insemination (BBIB) Singosari Malang. The method is laboratory research using completely randomized design. Consist of CEP, CEP + KT, CEP + PT, CEP + KT + PT and repeated 10 times. The variables are motility, viability and abnormality of sperm. Data were analyzed by using variance analysis. If the treatment effect then continued by Least Significant Difference (LSD). The results showed that the quality of Etawa cross-bred goat sperm with various diluents showed a significant influence (P <0.01). Motility and viability showed differences (P <0.01) in various diluents. The best motility and viability on top layer of CEP+KT+PT diluent were 65% and 69%, 55%. Percentage abnormalities showed that the treatment was not significant effect (P> 0.05). but top layer CEP+KT+PT diluent shows the percentage of abnormality west 4.88% compared to the other treatments. From the results of this study concluded that the quality of sexing semen quality by using dilution CEP + yolk white egg on the top layer gives the best results on the quality of sperm in terms of motility, viability and abnormalities. Based on this study it is suggested that the use of sexing semen with egg white sedimentation method using diluent CEP + yolk + white egg.
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Carvalho, J. O., R. Sartori, G. M. Machado, G. B. Mourão, and M. A. N. Dode. "365 QUALITY ASSESSMENT OF BOVINE CRYOPRESERVED SPERM AFTER SEXING BY FLOW CYTOMETRY AND ITS USE FOR IN VITRO EMBRYO PRODUCTION." Reproduction, Fertility and Development 22, no. 1 (2010): 339. http://dx.doi.org/10.1071/rdv22n1ab365.
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Several studies using sex-sorted sperm by flow cytometry have shown that its fertility is reduced. Therefore, this study evaluated structural and functional characteristics of sperm sexed by flow cytometry. In addition, in vitro embryo production (IVP) and development was assessed when frozen-thawed unsorted and sex-sorted sperm from 4 Nellore bulls. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). After thawing, each sample was analyzed for sperm motility by computer-assisted semen analysis (CASA, Berkeley, CA), sperm head agglutination, sperm morphology, membrane integrity by propidium iodide (PI) and 6-carboxy-fluorescein diacetate (CFDA) staining, acrosome integrity by peanut agglutinin (PNA), capacitation by chlortetracycline (CTC), and chromatin integrity by acridine orange staining. Then, the samples were placed in 45 : 90% (NS90) or 45 : 60% (NS60, SX, and SY) Percoll™ gradients. After Percoll™ centrifugation, sperm pellets were analyzed or used for IVP. All analyses were replicated independently three times. For IVP, 2,271 in vitro matured oocytes were used. To assess fertilization rate, presumptive zygotes were fixed and stained with lacmoid at 18 h post-insemination (hpi). Cleavage was evaluated at Day 2 (48 hpi) and blastocyst development at Days 6, 7, 8, and 9 of culture. Data were analyzed using generalized linear models. No differences (P > 0.05) were observed between SX and SY groups for e sperm variables evaluated either before or after Percoll™. However, non-sexed sperm had higher sperm motility, greater percentage of sperm with intact membranes, and greater percentage of live sperm with intact acrosomes than sexed sperm (P < 0.05). An effect of Percoll™ was observed in the non-sexed samples, with those submitted to 45 : 90% gradient having higher motility, greater percentage of cells with intact membrane, and lower recovery rate than those submitted to a 45 : 60% gradient. No differences among groups were observed for fertilization rate, being 74.0 ± 5.7, 63.2 ± 5.1, 67.2 ± 5.7, and 55.4 ± 5.9% for NS90, NS60, SX, and SY, respectively. Group NS90 showed a greater cleavage rate than did the SY group, while groups NS60 and SX had similar rates to the others. Blastocyst development rates on Day 6 to Day 9 were greater for group NS90. For example, on Day 8 the blastocyst rate was 34.9 ± 3.6, 22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9% forNS90, NS60, SX, and SY groups, respectively. All groups showed similar embryonic developmental stages on Day 6 to Day 9. Although sex-sorting affected sperm characteristics, it did not cause a decrease with in vitro fertility. However, differences in blastocyst rates between groups NS60 and NS90 indicated that the sperm selection protocol affected embryo production. Financial support: Embrapa Genetic Resources and Biotechnology.
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O'Brien, J. K., and T. R. Robeck. "Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)." Reproduction, Fertility and Development 18, no. 3 (2006): 319. http://dx.doi.org/10.1071/rd05108.
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Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 ± 4% purity) of X- and Y-bearing spermatozoa was 3400 ± 850 spermatozoa sex−1 s−1. In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen–thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.
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Richard, Kenny R., Steven W. McCarrey, and Jonathan M. Wright. "DNA sequence from the SRY gene of the sperm whale (Physeter macrocephalus) for use in molecular sexing." Canadian Journal of Zoology 72, no. 5 (May 1, 1994): 873–77. http://dx.doi.org/10.1139/z94-118.
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We sequenced a 152 base pair fragment of the sperm whale (Physeter macrocephalus) SRY gene in order to obtain species-specific primers for determination of sex by the polymerase chain reaction (PCR). Sequence identity between the sperm whale SRY fragment and the homologous motif in a variety of other mammals was high, though generally higher with ungulates (~88%) than with the human (85%), rabbit (80%), mouse (75%), or marsupial mouse (66%). When primers based on the sperm whale sequence were employed for PCR, a single product was amplified from male sperm whale DNA, whereas no product was amplified from female DNA. This SRY fragment was also amplified from other cetacean male DNA, but not from human DNA. Thus, under appropriate PCR conditions, the use of the cetacean-specific primers in an assay to determine sex eliminates the possibility of false results owing to contamination by human DNA. To confirm the results of the SRY analysis we sexed the same individual sperm whales by restriction analysis of PCR-amplified fragments from the ZFY and ZFX genes, using universal primers and methods previously reported to work for other cetacean species.