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Determination of the ratio of X and Y bearing spermatozoa after albumin gradient method using double-labelled fluorescence in situ hybridization (FISH).

Determination of X and Y human spermatozoa by double labelled fluorescence in situ hybridization (FISH) is a new and more reliable method than Quinacrine hydrochloride staining for Y sperm identification. A preliminary report on determination of the ratio of X and Y sperm of normal semen of fifteen donors by double labelled FISH method before and after Y sperm separation by Ericsson albumin column method. This is currently popularized in Thailand. Results of standardized FISH technique has an average success rate of approximately 95 percent. Results of the experiment showed that the ratio of X:Y sperm before and after albumin column filtration is 50.03:49.80 and 48.77:51.00, respectively. Few spermatozoa have double sex chromosomes, XX, XY or YY. In conclusion, Y sperm selection by Ericsson's method is ineffective for male sex preselection.

Cell Separation↗

In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer.

The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P<.05) were obtained when FERT-B was used as a fertilization medium for unsorted (43.4+/-5.1%) and sorted sperm (43.1+/-1.1%;), whereas in FERT-A unsorted sperm gave a cleavage rate of 17.9+/-4.4% and sorted sperm gave 30.4+/-1.4%. Additionally, cleavage rates were higher (P<.05) after fertilization with sorted sperm vs unsorted sperm, independent of fertilization medium. Cytogenetic analysis of ova revealed that more oocytes with unsorted than with sorted sperm remained in Metaphase 2 arrest (P<.05). This was also independent of the fertilization medium. Monospermic fertilization rates were the same for IVF with unsorted or sorted sperm, independent of the fertilization system, except FERT-A with unsorted sperm (P<.05). Polyspermic fertilization rates were highest in FERT-B (37.6+/-6.6). A total of 57 pigs were born from nine litters. Six litters from sexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.

Animals↗

Reproductive autonomy and evolutionary biology: a regulatory framework for trait-selection technologies.

The Constitution protects, in some measure, each person's autonomy in making basic decisions about family, parenthood, and procreation. This Article examines the extent to which courts should protect from government intrusions a parent's access to technologies that influence specific characteristics of offspring. Beginning with Supreme Court opinions that articulate constitutional and social values regarding reproductive autonomy, the Article explores how important new insights from evolutionary biology may supplement an understanding of human procreation. Specifically, the Article explains how trait selection can constitute an important part of larger "reproductive strategies" that powerfully affect an individual's "inclusive fitness" (itself a measure of reproductive success). It concludes that access to trait-selection technologies should receive the same federal protection from government intrusions as that afforded access to abortion. It proposes the first limit to that protection, however, when a parent seeks to select for a trait, or to use a technique, that would be clearly and significantly damaging to the future child. The Article subsequently divides the use of trait-selection technologies (TSTs) into eight contexts and proposes a preliminary framework by which a regulatory system could legitimately distinguish among them.

Biological Evolution↗

Gender preselection in mammals: an overview.

Recent advances in the separation of X and Y chromosome bearing spermatozoa have led to the availability of a method (Beltsville Sperm Sexing Technology) to preselect the sex in several mammals. Progeny using this procedure have been produced in cattle, sheep, swine and laboratory animals. Mammalian sperm are inherently different in that the X sperm carries from 2.8 to 7.5% more DNA than the Y sperm. Individual sperm DNA can be determined and used as the differentiating characteristic with flow cytometry and cell sorting instrumentation especially modified to measure small amounts of DNA in sperm. The process utilizes the fluorochrome Hoechst 33342 to bind to the DNA. The relative DNA is measured by passing the living sperm through a laser beam and collecting the light energy from the individual sperm. Data is acquired and used to select the particular sperm for deflection into collection tubes. The proportions of sorted X and Y sperm in each tube can be validated by reanalyzing an aliquot for DNA content. This value is then used to predict the outcome of fertilization and subsequent gestation. The sorted sperm are used to inseminate eggs via in vitro fertilization (IVF) or by surgical insemination into the oviduct or the uterus of appropriate females. Sperm are sorted at the rate of 0.5 million per hour for most species with the expectation of 90% or greater of one sex or the other being born. Progeny in cattle using IVF have been produced at greater than 90% accuracy. Rabbits have produced greater than 90% females using this process. Progeny produced from pigs average 85% for one sex or the other. All progeny produced (N = or > 300) have exhibited completely normal morphological appearance and normal reproductive function. Because of the inability to obtain large numbers of sorted sperm in a short amount of time, the technologies use for regular artificial insemination would not be practical in most domestic species. This sexing technology however is very applicable where IVF, intrauterine or intratubal insemination are convenient means for producing offspring. In addition, the recent advent of ultrasound guided insemination in cattle may provide and opportunity to use this technology for much lower numbers of sperm per insemination than previously thought possible. Using less than 2 x 10 sorted X or Y sperm would move the technology one step closer to practicality.

Animals↗

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio.

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.

Animals↗

Assisted reproductive practice: religious perspectives.

It is important to those who practise reproductive techniques to learn about different religious perspectives related to reproductive health problems. Religious groups are active in influencing the public regarding bioethical positions, and this is particularly evident with issues concerning procreation, abortion and infertility therapy. The Jewish attitude towards procreation is derived from the first commandment of God to Adam to 'Be fruitful and multiply'. Judaism allows the practice of all techniques of assisted reproduction when the oocyte and spermatozoon originate from the wife and husband respectively. The attitude toward reproductive practice varies among Christian groups. While assisted reproduction is not accepted by the Vatican, it may be practised by Protestant, Anglican and other denominations. According to traditional Christian views, beginning at conception, the embryo has moral status as a human being, and thus most assisted reproductive technologies are forbidden. According to Islam, the procedures of IVF and embryo transfer are acceptable, although they can be performed only for husband and wife. Developments in science and technology and corresponding clinical applications raise new religious questions, often without clear answers. The role of theology in bioethics is integral to clarify perceived attitudes toward these developments for different religious communities. This paper presents the attitude of monotheistic religions to therapeutic procedures, such as IVF-embryo transfer, spermatozoa, oocytes, embryo donation, cryopreservation of genetic material, surrogacy, posthumous reproduction, gender preselection, reproductive and therapeutic cloning.

Attitude↗