Fractionation of X and Y Chromosome-Bearing Bovine Spermatozoa through Sugar Gradients for Sex Predetermination in Dairy Cattle

Abstract

Abstract-Mankind has been interested in sex preselection since ancient times. It can be achieved either by sexing of early embryos or by separation of X and Y chromosome-bearing spermatozoa, but new separation techniques with better accuracy and low costs are necessary. The objective of this study was to fractionate X and Y-bearing bovine sperm using sucrose gradient. A discontinuous sucrose density gradient was prepared by layering successive decreasing sucrose solution upon one another. Finally 20μL semen sample was loaded on the top layer. Then it was centrifuged at 500 x g for 12 minutes at room temperature. After elution of fractions and centrifugation (at 700 x g for 5 minutes), sperm viability and acrosome integrity were assessed by using 0.4% Trypan Blue and 0.75% Giemsa stain. Other part of the pellet was stained with 2% orcein red for 30 minutes to obtain sets of chromosomes. Repeated measures analysis of variance (ANOVA) with Bonferroni's multiple comparison test was performed to compare the percentages of female sperms at every layer. Results have shown that means of percentage of X chromosomes increased from layer 1 (15.55 ± 2.939 %), layer 2 (14.0 ± 3.055%), layer 3(26.33 ± 0.881%) to layer 4 (31.85 ± 5.186), but there is a statistically significant difference between layer 2 and layer 4 (P<0.05). However it needs to perform further studies to obtain appropriate density gradient model. Our present study demonstrates that the discontinuous sucrose density gradients can be considered as low cost tool for sperm sexing of bovine semen.

Potensi Alfa Enolase (ENO1) Membran Plasma Spermatozoa Sapi Bali Sebagai protein Antigenik

“…Semen/ejakulat sampel penelitian ditampung dengan menggunakan vagina buatan (Kanesharatnam et al 2012). Pemeriksaan secara makroskopis yang meliputi volume, warna, konsistensi dan pH dilakukan setelah semen ditampung.…”

Tingkat keberhasilan Inseminasi Buatan (IB) menggunakan semen beku hasil sexing pada Sapi Persilangan Ongole

“…al., 2016). Spermatozoa X yang lebih berat akan mengendap dibagian bawah, sedangkan spermatozoa Y yang lebih ringan akan bermigrasi ke bagian atas (Kanesharatnam, Eswaramohan and Balasubramaniam, 2012).…”

Five Layered Optiprep based Density Gradient Model is a Promising Model for Enrichment of Viable X Chromosome Bearing Spermatozoa in Bubalus bubalis

“…Concerns have also been raised regarding Percoll's safety and its use in assisted reproductive technologies (Hossepian et al, 2000). Sucrose as a low cost alternative was used for density gradient media but with limited success Kanesharatnam et al (2012). Therefore, the primary objective of this study was to develop appropriate density gradient model for enriching viable X chromosome bearing spermatozoa population in semen of Murrah buffalo bull.…”