Enrichment of Bovine X and Y Spermatozoa by Free‐Flow Electrophoresis

Blöttner, S.; Bostedt, H.; Mewes, Karsten; Pitra, C.

doi:10.1111/j.1439-0442.1994.tb00113.x

Cited by 29 publications

(13 citation statements)

References 14 publications

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“…Apesar de Ishijima et al (1991) terem observado em seus experimentos que a coloração com quinacrina mustarda deu um erro de 16,4%, Blottner et al (1994) não observaram o mesmo quando compararam os resultados de seu experimento com aquele obtido por hibridização in situ.…”

Section: Resultsunclassified

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

Lima¹,

Moreira‐Filho

Lucio

et al. 2011

R. Bras. Zootec.

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RESUMO -O objetivo neste trabalho foi desenvolver um método de seleção do sexo de espermatozoides bovinos por centrifugação em gradiente de densidade de Percoll. Utilizou-se sêmen congelado de touros mantidos em regime de colheita de sêmen. A fração de espermatozoides X ou Y foi separada por centrifugação em treze diferentes gradientes de densidade de Percoll formados por 1 a 12 camadas com densidades que variaram de 1,004 g/mL a 1,123 g/mL. As soluções com diferentes densidades foram preparadas misturando-se, em proporções diferentes, meio de cultura Hank's e uma solução estoque composta de NaCl 1,5 M e Percoll (1:9, v/v). Sobre cada gradiente foi colocado um total de 50 × 10 6 espermatozoides descongelados em 0,7 mL de meio Hank's e centrifugados a 250 X g por 30 minutos, em rotor horizontal, a 25°C. Os espermatozoides das frações superior e inferior foram tratados com Quinacrina Mustarda e analisados (200 deles) quanto à presença do corpúsculo-F. Dos espermatozoides encontrados no sedimento de dois gradientes, compostos de 8 e 12 frações com densidades variando entre 1,050 a 1,120 g/mL e 1,044 a 1,123 g/mL, respectivamente, visualizaram-se 25% com corpúsculo-F e os 75% restantes prováveis portadores do cromossomo X. O aumento na porcentagem de espermatozoides X após a centrifugação em gradiente de densidade permitirá que esse método de sexagem seja usado em larga escala na produção comercial de carne e leite bem como no teste de progênie. Palavras-chave: Bos taurus, seleção do sexo, sêmenSex selection in bovine spermatozoa by using Percoll discontinuos density gradient centrifugation ABSTRACT -The objective of this work was to develop a bovine spermatozoid sex selection method by using Percoll density gradient centrifugation. It was used frozen semen of bulls kept in semen collection regime. Fraction X or Y was separated by centrifugation in three different Percoll density gradient formed by 1 to 12 layers with densities varying from 1.004 g/mL to 1.123 g/mL. Solutions with different densities were prepared by mixing, at different proportions, Hank's culture medium and a stock solution composed of NaCl 1.5 M and Percoll (1:9, v/v). On each gradient, it was put 50 × 10 6 spermatozoids thawed in Hank's medium and centrifuged at 250 X g for 30 min in a horizontal rotor, at 25°C. The spermatozoids in the superior and inferior fractions were treated with Mustard Quinacrine and analyzed (200 spermatozoids) for the presence of F-body. Of the spermatozoids found in the sediment of the two gradients, composed of 8 and 12fractions with densities varying from 1.050 to 1.120 g/mL and 1.044 to 1.123 g/mL, respectively, it was identified 25%with the F-body and the other 75% were probably X chromosome-bearing spermatozoids. Increase in percentage of the X-spermatozoid after density gradient centrifugation will allow this system of spermatozoid sexing to be used in large scale in commercial production of meat and milk as well as for progeny tests in bovines. IntroduçãoA sexagem de espermatozoides tem valor econômico signifi...

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Section: Resultsunclassified

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

Lima¹,

Moreira‐Filho

Lucio

et al. 2011

R. Bras. Zootec.

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“…Other authors also noted enrichment of spermatozoa by sex chromosome identification following free-flow electrophoresis, although this was variable (Shishito et al, 1975;Blottner et al, 1983;Kaneko et al, 1983Kaneko et al, , 1984Engelmann et al, 1988;Masuda et al, 1989;Blottner et al, 1994). It remains unclear whether such enrichment results from differences in surface charge or motility between X and Y chromosome-bearing spermatozoa, selective damage of one type of spermatozoa, or a combination of several factors.…”

Section: Discussionmentioning

confidence: 99%

Effect of sperm motility on separation of bovine X- and Y-bearing spermatozoa by means of free-flow electrophoresis

Manger¹,

Bostedt²,

Schill

et al. 2009

Andrologia

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Free-flow electrophoresis was used on bovine spermatozoa to test the hypothesis that there are surface charge differences between X and Y chromosome-bearing spermatozoa.Spermatozoa were deflected towards the anode by means of tromethamine (THAM) buffers of varying concentrations, and were separated into two populations under specific conditions. Experimental temperature and initial sperm motility had a significant effect on sperm distribution in the electric field. The results of DNA hybridization assays indicated an enrichment for Y chromosome-bearing spermatozoa.

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“…Twelve ejaculates from one Asian and three ejaculates (n = 2) from African elephants were collected by manual rectal stimulation (Schmitt and Hildebrandt 1998, 2000). Fresh samples from WR and BR bulls and spermatozoa recovered from the epididymidis were diluted in MES/HEPES semen extender (Blottner et al. 1994) to a concentration of 100 × 10 6 spermatozoa/ml for incubation at 37 or 15°C.…”

Section: Methodsmentioning

confidence: 99%

Germany/Australia Index of Sperm Sex Sortability in Elephants and Rhinoceros

Behr

Rath²,

Hildebrandt

et al. 2009

Reprod Domestic Animals

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Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

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Enrichment of Bovine X and Y Spermatozoa by Free‐Flow Electrophoresis

Cited by 29 publications

References 14 publications

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

Effect of sperm motility on separation of bovine X- and Y-bearing spermatozoa by means of free-flow electrophoresis

Germany/Australia Index of Sperm Sex Sortability in Elephants and Rhinoceros

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