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Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells. [ABSTRACT FROM AUTHOR]

Several approaches to sperm sexing such as Albumin gradient, Identification of H-Y antigen, Free-flow electrophoresis, Detection of sex-specific proteins, Centrifugal counter current distribution, Volumetric differences, Percoll density gradient, Flow-cytometry, Laser ablation, etc were put forward by the scientists. However, the alteration in DNA content between X- and Y-sperm remains the key and only discriminating feature for sperm sorting based on which only Flow cytometry and Laser ablation techniques are being used commercially. Flow cytometry is based on precise staining of the DNA of sperm with the nucleic acid-specific dye, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized highspeed sorter. Laser ablation (LA) is a system and method for sorting a mixture of stained particles in a fluid flow path. It includes an electromagnetic radiation source for exciting fluorescence emissions from the stained particles, a photodetector for detecting the fluorescence emissions, a processor for classifying the stained particles, and a photo-damaging laser for damaging selected particles in the flow path. [ABSTRACT FROM AUTHOR]

ABSTRACT. Knowledge of farmers about estrus detection is one of the factors that affect fixed-time artificial insemination (AI). This study aims to evaluate the success rate of AI using Y sexing frozen semen based on the estrus character of cattle. Forty-five Limousin Crossed Cows involved in this research with a Body Condition Score of 3-5 (1-9 scale), 1.8-7 years old. Y sexing frozen semen is produced by the Singosari Center for Artificial Insemination using Percoll's Gradient Density Centrifugation method. Observation of estrus character was done before insemination with the deep insemination technique of AI. Artificial insemination was carried out using double doses at the 2nd and 8th hours after estrus. Rheinbio vitamins as BioATP+ are injected after AI. The results showed that the Conception Rate and Pregnancy Rate based on the estrus character was higher as indicated by the red colour of the vulva was 17.78% and 22.22%; very swollen vulva was 20% and 22.22%; abundant cervical mucus was 15.56% and 17.78%, and vaginal temperatures ranging from 38.0-38.5oC was 13.33% and 20%. In conclusion, the low percentage of pregnancy in this study was caused by various factors, mainly the genetic quality, early embryonic efficacy, maintenance management especially feed, and there were cows that experienced reproductive disorders. (Evaluasi keberhasilan inseminasi buatan menggunakan semen beku sexing berdasarkan karakter estrus yang berbeda) ABSTRAK. Pengetahuan peternak mengenai deteksi estrus merupakan salah satu faktor yang berpengaruh terhadap ketepatan waktu inseminasi. Penelitian ini bertujuan untuk mengevaluasi tingkat keberhasilan IB menggunakan semen sexing Y berdasarkan karakter estrus ternak. Akseptor yang digunakan pada penelitian ini berjumlah 45 ekor Sapi Persilangan Limousin dengan BCS 3-5 dan umur berkisar antara 1,8-7 tahun. Semen beku sexing Y merupakan hasil produksi Balai Besar Inseminasi Buatan (BBIB) Singosari menggunakan metode Sentrifugasi Densitas Gradien Percoll (SGDP). Pengamatan karakter estrus dilakukan sebelum inseminasi. Inseminasi Buatan dilakukan dengan menggunakan double dosis pada jam ke-2 dan jam ke-8 dengan teknik deep insemination. Vitamin Rheinbio sebagai BioATP+ diinjeksikan setelah IB. Hasil penelitian menunjukkan bahwa keberhasilan kebuntingan ditinjau dari nilai conception rate (CR) dan pregnancy rate (PR) berdasarkan karakter estrus ternak lebih tinggi ditunjukkan pada kondisi warna vulva merah merata yaitu 17,78% dan 22,22%; vulva yang sangat bengkak yaitu 20% dan 22,22%; banyak mengeluarkan lendir servik yaitu 15,56% dan 17,78%, serta suhu vagina yang berkisar antara 38,0-38,5oC yaitu 13,33% dan 20%. Persentase kebuntingan yang rendah pada penelitian ini disebabkan oleh berbagai faktor, utamanya kualitas genetik ternak, kematian embrio dini, manajemen pemeliharaan khususnya pakan, dan terdapat ternak yang mengalami gangguan reproduksi.

ABSTRAK. Penelitian ini bertujuan mengevaluasi tingkat keberhasilan IB semen beku sexing Y. Penelitian menggunakan 39 sapi Peranakan Ongole dan 77 sapi Persilangan Limousin yang di IB secara double dosis. IB menggunakan semen beku spermatozoa Y dengan metode pemisahan Sentrifugasi Gradien Densitas Percoll (SGDP) sapi Limousin yang diproduksi oleh Balai Besar Inseminasi Buatan (BBIB) Singosari. Teknik deposisi semen adalah 4+ yaitu pada posisi cornua uteri selanjutnya dilakukan IB pada jam ke 2 dan ke 8. Untuk akseptor dilakukan penyuntikan BioATP+ dan pemberian pakan konsentrat 3 kg per hari selama 3 hari setelah IB. Variabel penelitian meliputi Non Return Rate (NRR), Service Per Conception (S/C), dan Conception Rate (CR). Hasil penelitian menunjukkan bahwa IB sapi Peranakan Ongole dan sapi Persilangan Limousin yaitu NRR1 sebesar 82,05% dan 89,61%, nilai NRR2 sebesar 76,92% dan 84,42%, nilai CR sebesar 58,97% dan 74,03%, serta nilai S/C sebesar 1,78% dan 1,46%. Kesimpulan penelitian adalah IB menggunakan semen beku hasil sexing Y sapi Limousin pada sapi persilangan Limousin memiliki tingkat keberhasilan yang lebih tinggi dibandingkan sapi Peranakan Ongole. (The success rate of artificial insemination using frozen sexing semen of different breed cow) ABSTRACT. This study aimed to evaluate the success rate of AI frozen semen sexing Y. The material in this study used 39 Filial Ongole and 77 Limousin crossbred cattle in AI in double dose. The spermatozoa used were Y frozen semen of Limousin bull with the Percoll Gradient Density Centrifugation (SGDP) separation method produced by the Singosari Center for Artificial Insemination. The semen deposition technique was 4+, which has the position of the cornua uteri (deep Insemination), then AI implemented with a double dose at the 2nd and 8th hours after estrous. The cow acceptor was injected with BioATP+ and feed by 3 kg concentrate per day for three days after AI. Research variables include Non-Return Rate (NRR), Service Per Conception (S/C), and Conception Rate (CR). The results showed that AI of Peranakan Ongol and Limousin crossbred cattle on NRR1, NRR2, CR values, and S/C values respectively was 82.05% and 89.61%; 76.92% and 84.42%; 58.97% and 74.03%; 1.78% and 1.46%. In conclusion, AI using frozen semen from sexing Y Limousin cattle in Limousin crossbred cows had a higher success rate than Filial Ongole cattle.

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The Artificial Insemination (AI)Â results in Beef cattle are expected to be male calf because they can increaseweight gain rapidly, so in this study AI was carried out by using sexing semen with Percoll density gradient centrifugation method containing Y sperm.This study was conducted to evaluate the success rate of AI using frozen sexing semen on Ongole Cross Breed. The study method used wasexperimental, comparing AI frozen sexing semen and frozen semen in 54 Ongole Cross Breed. The study variableswere Non Return Rate1 (NRR1), Non Return Rate2 (NRR2) and Conception Rate (CR).The study showed that the success rate of based on the value of NRR1 and NRR2AI using frozen sexing semen on Ongole Cross Breed is lower than un sexed frozen semen with the NRR1 value is 73,91%; 93,54% while the value of NRR2 was 69,56%; 83,87%. Based on CR value of frozen sexing semen higher than un sexed frozen semen showed CR valuewas 43,47%; 35,48%.

Abstract: Currently, there are few inexpensive, reliable, effective methods for commercially separating X- and Y-chromosome bearing fresh and frozen bovine sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlus™ (HP) which claims to alter the sex ratio in favor of female calves following artificial insemination. Three trials included the insemination of hyperstimulated cows with Control or HP-treated semen, non-surgical embryo collection on Day 7, and a combined PCR/dot blot assay to determine embryo sex. Chi-square analysis showed that the Control group produced a greater proportion (p <0.0005) of female embryos than the HP group. There were no differences in the proportions of transferable compared with degenerate embryos or in number of ovulations, embryos, and unfertilized ova collected from Control compared with HP groups. When treatments were combined, one of the two bulls used in the hyperstimulation studies produced an overall greater proportion of females (p <0.05), suggesting a bull effect. Another trial involved the insemination of cows synchronized via OvSynch® with fetal sexing via ultrasonography. Results of these studies indicated that HP semen sexing kit did not alter the sex ratio in favor of females in either hyperstimulated or single-ovulating cows; however, potential bull effects may be further evaluated to understand the capacity of HP with semen from specific bulls. Additionally, perhaps the sex of the surviving embryo can be manipulated by the maternal side, through ovarian, hormonal, oviductal, or uterine influences. [Copyright &y& Elsevier]

Abstract: The first objective was to correlate calving sex-ratio data from semen lots with the semen sex ratio obtained by two duplex polymerase chain reaction (PCR)/gel electrophoresis techniques. The two techniques involved different starting DNA amounts, PCR conditions, agarose gel concentrations, sample placement on the gels, lane size, number of lanes per gel, and duration of electrophoresis. The second objective was to sequence the duplex PCR products to verify their match to genes and chromosomes for which they were designed. Thirty-six ejaculates (lots) from eight Holstein sires were collected. Semen straws were distributed among dairies in three states. Ten straws per lot were used for the different PCR techniques. Sperm DNA was extracted and PCR analysis was done using one primer set to amplify a single copy section of the factor IX precursor (X-chromosome only) and another primer set to amplify a single copy section the sex determining region (Y-chromosome only). The glyceraldehyde phosphate dehydrogenase gene was amplified as an internal control. Standard curves were designed using PCR products in known ratios. Gel electrophoresis and image analysis were used to determine predicted %Y-chromosome-bearing spermatozoa (PredPtY). Sex (male=1, female=0) was reported on 526 calves and the ratio of the number of male to total calves (proportion of male calves (PMC)) was determined between sire and lot within sire. The PredPtY and PMC were significantly correlated (r =0.82, P <0.0002). No significant variance between sires was found in PredPtY or PMC, but lots within sires was a significant variance source for both. The two PCR technologies adequately determined semen sex ratio. The technology-by-lot-within-sire interaction was a significant variance source for PredPtY. Acrosomal integrity (after a 2-h) incubation, was correlated with both PMC and PredPtY; other semen quality characteristics had no significant correlations with PMC or PredPtY. Therefore, calf crop sex ratio skewness could be controlled by screening semen for PredPtY through the use of PCR. [Copyright &y& Elsevier]