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PubMed2006

Sex ratio determination in bovine semen: a new approach by quantitative real time PCR.

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.

Animals↗
PubMed1986

Sex chromosome ratios determined by karyotypic analysis in albumin-isolated human sperm.

Human sperm that had been processed for Y-enrichment (male sex preselection) according to a currently favored albumin density gradient procedure were analyzed karyotypically for the proportion of X and Y chromosomes with the use of the human sperm/hamster egg system. This method allows direct inspection of haploid chromosome complements from human sperm. In 290 albumin-isolated sperm from six men, there were 57.2% X- and 42.8% Y-bearing chromosome complements; 201 unprocessed concurrent control sperm from five of the men had 50.2% X and 49.8% Y complements. The observed shift in sex chromosome ratio in processed samples, a decrease in Y-bearing sperm, was not statistically different from that of unprocessed controls (P = 0.13) but was significantly different when compared with the theoretic X/Y ratio of 50/50 (P = 0.016). A total of 3187 historical control karyotypes were also reviewed, with an overall sex chromosome ratio (X/Y) of 49.8/50.2. The control groups did not differ significantly from the expected 50/50. The Y-enrichment of processed sperm was not confirmed.

Animals↗
PubMed2000

X-Y sperm selection: fact or fiction?

Selecting the gender of offspring has given rise to various and sometimes amusing stories. But regardless of which prefertilisation technique is used to influence the sex ratio of offspring it must fulfill certain criteria. First of all it must achieve a complete separation of the X and Y bearing sperm in sufficient quantities. Secondly sperm must be viable after separation and capable of fertilising. Sex preselection methods can be divided into two general groups which either separate spermatozoa on the basis of subtle physical or kinetic features or those which rely on distinctive nuclear characteristics unique either to X or Y chromosome bearing sperm. These, in turn, can be divided into in vivo methods designed to produce optimal conditions for fertilisation by either the X or Y bearing sperm, or in vitro sperm separation methods designed to separate X or Y bearing sperm. According to all published data, the different separation techniques have been shown not to be very effective. Only sex selection of spermatozoa by chromatin differences (cell sorting by flow cytometry) has demonstrated a significant enrichment of the X bearing sperm.

Chromosomes, Human, X↗
PubMed1989

[The sex ratio at birth: a retrospective review and commentary].

Trends in the sex ratio are examined, and the possible effect of new biotechnologies on sex preselection is discussed. "We recall that this ratio is very stable, around 105 males for 100 females (live births). However, in France, a slight decreasing trend during the 19th century can be observed (from 107 to 104). At the present time this ratio seems to be maintained at a level of 105.3. The great demographic perturbations, particularly caused by wars, have been marked by a slight but notable increase in the indicator (106) when natality rises again after a deep depression. The variations, always small, of this indicator are more dependent on the male than on female natality, for reasons that are unclear." A comment by Paul Damiani is included (pp. 99-102). (SUMMARY IN ENG)

Biology↗
PubMedDate not supplied

Fetal sex preference of second-trimester gravidas.

Fetal sex preferences of pregnant women are based on psychological, physiologic, economic, and sociologic factors. Parental responses to the knowledge of fetal/infant sex range from feticide to preferential treatment of the preferred-sexed child. This study describes the sex preferences, sex beliefs, attempted sex preselection techniques, and desire to know the fetal sex of 243 second-trimester gravidas. Data demonstrated a willingness to disclose sex preferences, with 81% declaring a preference. The majority of women (81%) also wished to know the sex of their child prior to delivery regardless of their acknowledgment of a sex preference. The results of this survey offer implications for research on the phenomena relating to sex preference.

Adolescent↗
PubMed1997

Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry.

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Journal Article↗
PubMed1982

Women's sex preferences in the United States: 1970 to 1975.

This analysis examines the potential effect of sex preselection technology in the United States. The results suggest that controlling the sex of offspring is not the desire of most American women; that if it were employed, there would be a significant increase in sons as first-born and daughters as second children; that the overall sex ratio would be little changed from that occurring naturally except at very low fertility levels with universal use of such technology; and that fertility is only minimally influenced by gender preferences.

Attitude↗
PubMed2004

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting.

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.

Analysis of Variance↗
PubMed1985

Preference for sex of child among primiparous women.

Primiparous American women (N = 140) were questioned during their third trimester concerning their choice of sex of offspring and their willingness to use sex preselection techniques (if available). Eighty-two women expressed no preference for sex of offspring; of the remaining 58, 33 chose girls and 25 chose boys. Of the 26 women who indicated they would have used preselection technology, 13 chose boys and 13 chose girls. Fifty-three percent of the sample indicated they would not have used preselection techniques; 29% were undecided. These results were at variance with earlier studies that indicated a consistent choice of male firstborns, especially in nonpregnant samples. The discrepancy is discussed in terms of a move from boy preference to no preference and a gradual weakening of societal bias against women.

Attitude↗
PubMed2002

Gender selection: cultural and religious perspectives.

OBJECTIVE: To review the current developments in the field of preconceptual sex selection and to discuss the cultural and religious perspectives as that accompany the scientific progress. DESIGN: A survey of the major publications in Judaism, Christianity and Islam regarding the issue of gender selection. Examination of current methods of preconceptual gender selection revealed that in vivo methods such as timing of intercourse, the use of ovulation induction medications, and artificial insemination do not appear to affect the sex ratio to a clinically significant degree. In vitro separation of X- and Y-bearing spermatozoa by gradient techniques have been reported to alter significantly the sex ratio at birth. However, these trials were not controlled, and molecular biological techniques could not validate that these methods indeed change the Y- to X bearing spermatozoa ratio sufficiently for clinical use. Nevertheless recent scientific advances have made highly reliable preconceptual sex selection possible by using preimplantation diagnosis (PGD) or sperm separation by flow cytometry combined with AIH or IVF. At present, these methods have been used to avoid sex-linked disorders. Both involve the invasive procedure of IVF and thus are held by most as inappropriate for nonmedical indications. However, improvement in flow cytometry output of sexed spermatozoa might provide in the near future sufficient sorted gametes for artificial insemination. It may be that in the near future, an improvement in flow cytometry output of sexed spermatozoa will provide sufficient sorted gametes for artificial insemination. In such a case, the medical community will be forced to take a stand, whether this reliable noninvasive method of sexing will be allowed for social purposes and even if the practice of PGD should be allowed for nonmedical indications. CONCLUSION: The requirement for a man to procreate by having a minimum of two children-a boy and a girl-is obligatory according to Jewish law. According to both schools, Beit Shamai and Beit Hillel, in order to fulfill the obligation of procreation at least one son is required. Therefore the application of sex preselection for nonmedical indications may by of practical importance using the method of sperm separation or sex selection of pre-embryo by PGD. According to Christian view, especially the one of the Catholic Church, gender preselection even for medical indications is forbidden. Islamic legal viewpoint is that fetal sex selection is lawful when it is practiced on an individual basis, to fulfill the wish of a married couple to have a boy or a girl through available medical means.

Christianity↗
PubMed2002

Gender selection: ethical, scientific, legal, and practical issues.

PURPOSE: This work offers an overview of the technology available and assisted reproductive technology (ART) practitioner attitudes to human reproductive sex preselection in either sperm with the use of flow cytometry (FC) or in embryos by means of preimplantational genetic diagnose (PGD) together with an analysis of Spanish legal environment. METHODS: A review of the legal, ethical and technical literature of the methods to select the sex in the offspring is performed. RESULTS: Sex selection in humans has different utilities to be employed depending on each country's law. Moreover, different ethical concerns are raised depending on the type of sex selection, in sperm or embryos. Both methodologies to pre-select the sex are trustworthy, with a high predictive power in the determination of the sex, but nevertheless, PGD is better than FC selection in this aspect. CONCLUSIONS: After a careful analysis of ethical, legal, and scientific features of gender selection, FC combined with PGD appears to be the most acceptable way to select the sex of the progeny whatever the circumstances are.

Female↗
PubMed2000

Sexing mammalian sperm for production of offspring: the state-of-the-art.

Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.

Animals↗
PubMed1999

Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads.

We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7-8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate.

Animals↗
PubMedDate not supplied

A note on the state of the art of preselection of the sex of children.

"In this paper the author presents the state of the art of preselection of the sex of children. To this purpose attempts to control the sex of offspring are classified into four categories: 1) selective conception; 2) treatment of ova or embryos, 3) presexing of embryos; and 4) selection of sperma. The nature and the availability of the different approaches are described as well as the various socio-medical aspects involved. In spite of the general impression that, at present, no reliable technique with clearly known probabilities of success and failure is available, this goal seems to be at close reach and sex selection of children will certainly become accessible in a not very remote future." (SUMMARY IN FRE AND ITA)

Adolescent↗
PubMed2004

Assessment of bovine X- and Y-bearing spermatozoa in fractions by discontinuous percoll gradients with rapid fluorescence in situ hybridization.

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 Y-bearing 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 Y-bearing 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.

Animals↗
PubMed1996

Analysis of the flow cytometer stain Hoechst 33342 on human spermatozoa.

Several procedures exist for processing sperm cells for sex preselection. Flow cytometric separation using the fluorochrome stain Hoechst 33342, chemically known as bisbenzimide, is the most promising. The objective of this study was to determine the effect of bisbenzimide on spermatozoa assessed by means of the sperm survival test and to analyse the beta-globin gene in sperm DNA after exposure to increasing concentrations of bisbenzimide. Donor (n = 16) sperm specimens were pooled and washed in a discontinuous Percoll gradient 95:47%, divided and incubated in tubes containing bisbenzimide at concentrations 0 (control), 0.9, 9, 90, 900 and 9000 microM at 25 degrees C and scanned on a computer-aided sperm motility analyser at 0, 1, 4 and 24 h. Spermatozoa were also incubated in a known mutagen, ethidium bromide, as positive control. After 24 h of incubation, the treated sperm cells were processed through DNA extraction and polymerase chain reaction (PCR) performed with primers targeting the beta-globin gene. The amplified DNA products were analysed for evidence of mutation in 5% polyacrylamide gel electrophoresis and 20:80 denaturing gradient gel electrophoresis (DGGE) and further confirmed in 30:40 DGGE. The results showed complete cessation of motility in sperm incubated in the presence of 900 microM or higher concentrations of bisbenzimide. The beat cross frequency sperm parameter was significantly different at the 90 microM or higher concentration of bisbenzimide compared with the control. At concentrations < 900 microM bisbenzimide, there were no differences in the remaining sperm kinematic parameters (percentage rapid progressive, percentage total progressive, sperm velocities, linearity, straightness, amplitude of lateral head displacement and percentage hyperactive motility). PCR and DGGE analyses of spermatozoa treated with bisbenzimide showed no evidence of mutation in the representative region of the beta-globin gene at concentrations < 900 microM. The data suggest an inhibitory effect of bisbenzimide on human sperm motility at 900 microM or higher concentrations of bisbenzimide. The decrease in sperm motility and rapid progression were not due to changes in pH. Point mutation in the representative region of the beta-globin gene in human spermatozoa was detected only at high concentrations (> or = 900 microM) of bisbenzimide. The data suggest that incubating sperm in low concentrations of bisbenzimide (< 90 microM) for up to 24 h does not significantly affect all the sperm kinematic parameters including the beat cross frequency parameter when compared with the control.

Benzimidazoles↗
PubMed1977

[Antenatal diagnosis of fetal sex. Method and implications].

An outline of fetal antenatal sex determination and its diagnostic methods are rapidly enumerated. Actual methods are based on direct and indirect identification of sexual chromosomes from three types of cells : desquamated ones in amniotic fluid, fetal lymphocytes circulating in maternal blood and desquamated chorionic cells from endocervical smear. Three methods are utilizable : evidence of sexual chromatine, fluorescence specificity of the Y chromosome and the karyotype. Cells from amniotic fluid permit a correct prediction up to 97,5 p.100. However, the high risk of contamination limits its use. There is a 86 p. 100 diagnostic certainty with fetal lymphocytes in maternal blood or cervical desquamated chorionic cells. Future sex preselection may be contemplated and along with this the speculative social consequences. On the other hand, sex determination and volontary selection are limited at the moment by available technics and also by scientific and medical ethics.

Amniocentesis↗
PubMed1981

Determining the impact of sex preferences on fertility: a demonstration study.

The stopping rule measure of sex preferences represents a combination of psychological measures of preference and behavioral intentions. This study of 172 college students demonstrates that the stopping rule measure is a useful and practical method of measuring sex preferences. The results further indicate that parity progression ratio measures inherently underestimate the effect of sex preferences on individual fertility because they incorrectly assume that sex preferences (a) are homogeneous within the population and (b) can only act to increase, not to decrease, fertility. Use of the stopping rule measure to predict the possible effects of sex preselection techniques on fertility is also discussed.

Decision Making↗