Improved flow cytometric sorting of X- and Y-chromosome bearing sperm: Substantial increase in yield of sexed semen

Rens, W.; Welch, G. Rickey; Johnson, Lawrence A.

doi:10.1002/(sici)1098-2795(199901)52:1<50::aid-mrd7>3.0.co;2-h

Cited by 44 publications

(28 citation statements)

References 16 publications

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“…This is in contrast to previous reports of delayed embryonic development after IVF with spermatozoa sorted on either a high speed (Lu et al 1999) or a standard speed flow cytometer (Cran et al 1993, Merton et al 1997 prior to freeze-thawing compared with non-sorted, frozen -thawed or fresh bull spermatozoa. Significant improvements in both biological (bull: Schenk et al 1999, ram: F K Hollinshead, unpublished observations) and instrumental (Rens et al 1999, Campos-Chillon & De La Torre 2003, Suh & Schenk 2003 aspects of flow cytometric sperm sorting have occurred since these reports. Such developments have significantly reduced the dilution rate of spermatozoa during staining (Maxwell & Johnson 1999) and may be contributing to improvements in the IVF parameters found in this study compared with earlier reports.…”

Section: Discussionmentioning

confidence: 99%

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

Hollinshead

Evans

et al. 2004

Reproduction

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The characteristics and functional capacity of ram spermatozoa frozen-thawed prior to and after flow cytometric sorting was assessed after incubation (37 8C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X-and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 6 1.5% and Y: 87 6 1.1% purity). After 6 h incubation (37 8C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 6 2.0%) compared with all other treatments (Control: 36 6 3.3%, FSF: 28 6 3.1%, FCF: 20 6 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 6 39.4 vs 31 6 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X-and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively).Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen-thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.

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Section: Discussionmentioning

confidence: 99%

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

Hollinshead

Evans

et al. 2004

Reproduction

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“…Special applications include for example sex pre‐selection in farm animal, using a modified high‐speed flow cytometry technique, which enables the shape‐depended alignment of sperm for quantitatively sorting into X‐ and Y‐chromosome bearing populations (Johnson et al. 1989; Rens et al. 1999).…”

Section: Nano‐revolution Of Genetics and Reproductive Biologymentioning

confidence: 99%

Golden Perspective: Application of Laser‐Generated Gold Nanoparticle Conjugates in Reproductive Biology

Barchanski

Taylor

Klein

et al. 2011

Reprod Domestic Animals

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Contents The current demand for female calves has grown rapidly and controlling the sex of offspring provides an economically flexible management for the livestock producer. The only functioning method of efficiently producing separate populations of X and Y sperm in mammals is based on relative DNA differentiation by high‐speed flow cytometry. In this context, gold nanoparticles conjugated to sex chromosome‐specific moieties display promising application as novel fluorophor‐alternative for the high‐throughput screening, since they feature no photo bleaching, high quantum yield, good biocompatibility and the possibility of non‐destructive membrane penetration. Especially, gold nanoparticles fabricated by pulsed laser ablation are in the recent focus of interest, due to excellent biocompatibility, fabrication‐dependent, tuneable particle size as well as surface charge and ease of (bio)‐functionalization with a remarkably strong ligand binding. For the purpose of our studies functionalized gold nanoparticles may be used as novel markers for sex‐sorting of mammalian sperm and, depending on the selected probe, also for the selection of sperm with heritable DNA‐sequences interesting for animal breeding.

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“…Initially, sort rates for sperm were approximately 80-90 sperm s -1 of each sex because only about 15 to 20% of intact sperm flowing past the laser beam of the instrument could be properly oriented for measurement Cran et al 1995Cran et al , 1997. Development of high-speed sorting systems ( Van den Engh and Stokdijk 1989), subsequently commercialized by DAKO-Cytomation, Inc. (Fort Collins, CO) plus modification of the sorter nozzle (Rens et al 1998(Rens et al , 1999 produced much higher sort rates Johnson 2000;Garner 2001). Implementation of low dose insemination protocols made this system capable of providing reasonable numbers of sperm for artificial insemination (Seidel et al 1997).…”

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confidence: 99%

Past, present and future perspectives on sexing sperm

Garner¹,

Seidel²

2003

Can. J. Anim. Sci.

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. 2003. Past, present and future perspectives on sexing sperm. Can. J. Anim. Sci. 83: 375-384. 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. Cette technologie, qui a été commercialisée récemment pour les spermatozoïdes de bovins, est basée sur les différences entre le contenu en ADN des chromosomes X et Y du spermatozoïde. Occasionnellement, sous des conditions exceptionnelles, 5 000 ou plus des spermatozoïdes bovins vivants, de chaque sexe peuvent être trié avec une exactitude de 90%. Avec le tri des spermatozoïdes de bovins cryoconservés, un taux de gestation de 50% a été réalisé couramment chez des génisses de fermes d'élites, comparé à 60-80% avec des spermatozoïdes non-triés. Environ 90% des progénitures ont été du sexe sélectionné. Il est possible de trier les spermatozoïdes d'une variété d'autres mammifères incluant les espèces exotiques. Quoi qu'il en soit, les taux, la pureté des tris et la viabilité des spermatozoïdes triés ne sont généralement pas aussi élevés que chez les spermatozoïdes de bovins.

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Improved flow cytometric sorting of X- and Y-chromosome bearing sperm: Substantial increase in yield of sexed semen

Cited by 44 publications

References 16 publications

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

Golden Perspective: Application of Laser‐Generated Gold Nanoparticle Conjugates in Reproductive Biology

Past, present and future perspectives on sexing sperm

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