Assessment of Bovine X- and Y-bearing Spermatozoa in Fractions by Discontinuous Percoll Gradients with Rapid Fluorescence In Situ Hybridization

Abstract

Abstract. This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X-or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Ybearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X-and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X-and Ybearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X-and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied. Key words: Percoll separation, Semen sexing, FISH, Spermatozoa, Bovine (J. Reprod. Dev. 50: [463][464][465][466][467][468][469] 2004) ince the 1970s, several investigators have made a tt e m p t s t o s e p a r at e X -a n d Y -b e ar in g spermatozoa by means of various gradient techniques such as discontinuous albumin gradients [1, 2], Shepadex column s [2,3], discontinuous Percoll gradients [4,5] and the swim-up procedure [6]. None of these methods, however, have met with success sustainable and reproducible enough to achieve separation of Xand Y-bearing spermatozoa, and only flow cytometry has been proven to be effective [7]. One o f t h e p r o b l e m s a s s o c i a t e d w i t h r e s e a r c h attempting the separation of X-and Y-bearing spermatozoa has been the difficulty of validating the proportion of X-and Y-bearing spermatozoa within putatively separated sperm populations [8]. Early studies evaluated the efficiency of sperm separation by using quinacrine staining. Recent evidence has shown that the quinacrine method is unreliable and may not necessarily detect only the Y body at the Y chromosome in spermatozoa [9].Current development of DNA probes raised against the sex chromosome specific sequence gives

Practical use of percoll density gradient centrifugation on sperm sex determination in commercial dairy farm in Thailand

“…This result was accordance with some previous findings (Kobayashi et al, 2004) that using multilayer Percoll ® gradients which could successfully enrich X-bearing bovine sperm.…”

“…Sperm preparation by 7-layer Percoll ® gradient was developed by Kobayashi et al (2004). A modified discontinuous 7 layer technique was used for the Percoll ® separation method in order to obtain an isotonic medium upon dilution with 100% Percoll ® .…”

“…This method has been successfully applied in human without deleterious effect (Kaneko et al, 1983). Moreover, this 2 layers method has been tested in bovine and swine (Kobayashi et al, 2004;Wolf et al, 2008). In this study, we demonstrated an extension of Percoll ® Density Gradient method to use as a simple sperm selection tool which is affordable for dairy farm industry.…”

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

“…In bovine, one centrifugation on 12-layer discontinuous Percoll gradient produced a female sex ratio deviation that ranged from 55.7 to 74.3% (Hossepian de Lima et al, 2000;Kobayashi et al, 2004). In the present study, the discontinuous density gradient with only 3 layers was converted into continuous density gradient and Arq.…”

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

“…The discontinuous Percoll gradient made with 12 layers can be used for sexing in bovines [9], [10]. In our study the discontinuous density gradient with only 4 layers was prepared for sexing.…”