Sperm Sorting Research Articles

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after “in vitro” capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.

Screening of sperm velocity by fluid mechanical characteristics of a cyclo-olefin polymer microfluidic sperm-sorting device

The microfluidic sperm-sorting (MFSS) device is a promising advancement for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection. However, these disposable devices were not clinically suitable for assisted reproduction, so a cyclo-olefin polymer MFSS (COP-MFSS) device was developed. By micromachining, two microfluidic channels with different heights and widths (chip A: 0.3 × 0.5 mm; chip B: 0.1 × 0.6 mm) were prepared. Sorted sperm concentrations were similar in both microfluidic channels. Linear-velocity distribution using the microfluidic channel of chip B was higher than that of chip A. Using confocal fluorescence microscopy, it was found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than in the middle because of the low fluid velocity. These results experimentally demonstrated that the width of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity. Microfluidic sperm sorting (MFSS) is a promising advance for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection; however, these devices are neither disposable nor clinically suitable for assisted reproduction, so we developed a cyclo-olefin polymer (COP)-MFSS device. By micromachining, we prepared two microfluidic channels with different heights and widths (chip A: 0.3 × 0.5 mm; chip B: 0.1 × 0.6 mm), and subsequently used these channels to investigate the sperm linear velocity distribution and the sorted motile sperm concentration. The sorted sperm concentrations were similar in both microfluidic channels. The linear velocity distribution using the microfluidic channel of chip B was higher than that of chip A. To investigate the activity of sorted motile spermatozoa at different heights of the microfluidic channel (top, middle and bottom), we performed confocal fluorescence microscopy and found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than at the middle of the channels because of the low fluid velocity. We experimentally demonstrated that the width, and not the height, of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity.

Transcriptional dynamics of the sex chromosomes and the search for offspring sex-specific antigens in sperm

The ability to pre-select offspring sex via separation of X- and Y-bearing sperm would have profound ramifications for the animal husbandry industry. No fully satisfactory method is as yet available for any species, although flow sorting is commercially viable for cattle. The discovery of antigens that distinguish X- and Y-bearing sperm, i.e. offspring sex-specific antigens (OSSAs), would allow for batched immunological separation of sperm and thus enable a safer, more widely applicable and high-throughput means of sperm sorting. This review addresses the basic processes of spermatogenesis that have complicated the search for OSSAs, in particular the syncytial development of male germ cells, and the transcriptional dynamics of the sex chromosomes during and after meiosis. We survey the various approaches taken to discover OSSA and propose that a whole-genome transcriptional approach to the problem is the most promising avenue for future research in the field.

Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one?

Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by “sort reanalysis” of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen.

Characterization of Whole, Single and Double Strand DNA Damage in Sex-Sorted Bovine Spermatozoa.

The objective of the present investigation was to examine the dynamics of DNA damage after flow cytometry sex-sorted bovine sperm. The hypothesis tested was that during sperm sorting, the level of DNA damage in the sperm fraction to be used for insemination can be reduced due to a step included in the methodology that removes non viable sperm although the DNA quality could be compromised. Eight sperm samples were sex-sorted using a MoFlo SX (Beckman Coulter, Miami, FL) to produce purified sperm subpopulations bearing the X or Y chromosomes. These samples and conventional non sorted sperm from the same ejaculates were cryopreserved. Sperm DNA fragmentation (SDF) of each sample was assessed after thawing. Evaluations occurred immediately after thawing, (t0), 24 and 48 hours of incubation at 37°C using Halomax (Halotech SL, Madrid, Spain). SDF at t0 was lower in sex-sorted samples, and statistical differences were obtained after comparing both groups (P 0.01). However, a reduction in DNA longevity was observed in sorted sperm during the 24 and 48 hour incubation periods. In these samples, SDF increased between 24 and 48 hours of incubation while in conventional samples this tendency was delayed. The graphic representation of SDF showed a crossover point between both dynamic tendencies, the Crossover Positioning Time (CPT). This CPT can be used as an indicator of sperm quality for each bull, suggesting that higher values of CPT are indicative of bulls with more resistant chromatin to external stressors that affect DNA after sorting, while those exhibiting lower values may have a compromised chromatin structure. The increase in SDF in sorted samples was characterized to gain information about the type of DNA damage (single strand break -SSB- or double strand break -DSB-) using a 2D-comet assay. The SDF after thawing was a mixture of SSB and DSB. However, DNA damage produced during incubation was characterized as SSB. Differences in the kinetics for DSB production after a 24h incubation time period were obtained when the different individuals were compared (Kaplan-Meier estimator -log rank test statistic- Mantel-Cox; χ2 6.12; P: 0001). The conclusion to this research is that sex sorting sperm decreases the initial sperm DNA damage when compared to conventional semen samples, but it increases the rate of sperm DNA damage over time. The iatrogenic DNA damage produced is mainly SSB. Since the oocyte has a superb capacity for DNA repair, could be of interest to investigate which are the threshold levels of SSB that may induce lethal and sub-lethal effects in the progeny, as well as those which could be repaired by the female gamete. (poster)

Effects of sex-sorting and sperm dosage on conception rates of Holstein heifers: Is comparable fertility of sex-sorted and conventional semen plausible?

The conception rates of Holstein heifers after AI with 2.1 or 10 × 106 sperm dosages of sex-sorted or conventionally processed sperm were compared. Ejaculates collected by artificial vagina from 8 Holstein sires were cryopreserved at either 2.1 or 10 × 106 sperm per dose with or without sorting to 90% purity for X-chromosome-bearing spermatozoa using flow cytometry. All treatments were processed in an egg-yolk (20%), TRIS, glycerol (7%) extender and packaged in color-coded 0.25-mL French straws. Straws (n=350 straws/treatment per sire) were packaged and distributed in aliquots of 12 (3 straws of each treatment) to 51 herds of Holstein heifers. Straw color was recorded in the on-farm record keeping system at the time of AI and retrieved by electronic download. In total, 9,172 services were recovered, providing a mean sample size of 287±3.5 services/sperm dose per semen type within sire (range: 248 to 318). Conception rates were influenced by the main effects of herd, sire, semen type, sperm dosage, and service number. The herd by sperm dosage interaction was the only interaction determined to be significant and implies that some herds (technicians) are more proficient than others at maintaining high levels of conception with decreased sperm dosages. Across herds and sires, the conception rates of each semen type by sperm dosage combination were as follows: 2.1 × 106 sex-sorted, 38%, n=2,319; 10 × 106 sex-sorted, 44%, n=2,279; 2.1 × 106 conventional, 55%, n=2,282; and 10 × 106 conventional, 60%, n=2,292. The observation that conception rates of sex-sorted semen were improved by the 10 × 106 sperm dosage is encouraging toward the prospectus of development of a commercially available sex-sorted product with improved conception potential over existing technology. However, the failure of the 10 × 106 sex-sorted sperm dosage to achieve conception rates comparable to either dosage of conventional semen is somewhat discouraging toward the plausibility of comparable conception rates to conventional semen in the absence of major technological advances in efficiency of sperm sorting or cryopreservation.

Natural levels of polyandry: differential sperm storage and temporal changes in sperm competition intensity in wild yellow dung flies

Summary 1. Polyandry is common in insects. Nevertheless, the evolutionary causes and consequences of this phenomenon remain contentious, in part because of a lack of information about natural mating rates and the fact that most post-copulatory processes are hidden from view within female reproductive tracts. 2. We captured wild female yellow dung flies (Scathophaga stercoraria) over the whole spring season and genotyped the sperm from their spermathecae to obtain information on sperm transfer, sperm storage and natural levels of polyandry for this model species of post-copulatory sexual selection research. 3. On average, females stored sperm from a minimum of 2·47 males (based on the most conservative estimate). Incorporating knowledge of population allele frequencies yielded a slightly higher estimate of 3·33 mates per female. 4. Sperm storage and therefore sperm competition intensity showed high temporal variation. The proportion of multiply mated females (i.e. females with sperm from ≥2 males within their sperm stores) and the absolute number of ejaculates detected within females increased strongly over the spring season before sharply decreasing as midsummer approached. 5. Interestingly, we detected a positive relationship between the number of stored ejaculates and females’ wing injuries, suggesting that mating not only causes measurable cumulative damage to wild females but also provides a potential mechanism by which males may be able to assess the intensity of sperm competition within a female. 6. Our study found no evidence for intraejaculate sperm sorting, but importantly, the number of ejaculates in storage differed amongst the three sperm storage organs (spermathecae) of female yellow dung flies. Different sperm mixtures across the spermathecae could enable females to bias paternity towards certain males if females can selectively use sperm from a certain spermatheca at the time of fertilization.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art.

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

Lensless imaging for simultaneous microfluidic sperm monitoring and sorting

5.3 million American couples of reproductive age (9%) are affected by infertility, among which male factors account for up to 50% of cases, which necessitates the identification of parameters defining sperm quality, including sperm count and motility. In vitro fertilization (IVF) with or without intra cytoplasmic sperm injection (ICSI) has become the most widely used assisted reproductive technology (ART) in modern clinical practice to overcome male infertility challenges. One of the obstacles of IVF and ICSI lies in identifying and isolating the most motile and presumably healthiest sperm from semen samples that have low sperm counts (oligozoospermia) and/or low sperm motility (oligospermaesthenia). Microfluidic systems have shown potential to sort sperm with flow systems. However, the small field of view (FOV) of conventional microscopes commonly used to image sperm motion presents challenges in tracking a large number of sperm cells simultaneously. To address this challenge, we have integrated a lensless charge-coupled device (CCD) with a microfluidic chip to enable wide FOV and automatic recording as the sperm move inside a microfluidic channel. The integrated system enables the sorting and tracking of a population of sperm that have been placed in a microfluidic channel. This channel can be monitored in both horizontal and vertical configuration similar to a swim-up column method used clinically. Sperm motilities can be quantified by tracing the shadow paths for individual sperm. Moreover, as the sperm are sorted by swimming from the inlet towards the outlet of a microfluidic channel, motile sperm that reach the outlet can be extracted from the channel at the end of the process. This technology can lead to methods to evaluate each sperm individually in terms of motility response in a wide field of view, which could prove especially useful, when working with oligozoospermic or oligospermaesthenic samples, in which the most motile sperm need to be isolated from a pool of small number of sperm.

20 FACTORS THAT AFFECT PURITY AND YIELD OF BOVINE SEX-SORTED SPERM

The purposes of these studies were to examine the influence on the yield of sex-sorted sperm by the different size of sorting gate and to evaluate factors that affect the purity of sex-sorted sperm. As the sorting gate was expanded, so the yield of bovine sex-sorted sperm using flow cytometry was increased. At the same time, the purity of sex-sorted sperm became low. In addition, though the sorting gates were the same size, the purity of sex-sorted sperm differed among trials. These observations indicated the existence of factors that affect purity besides the size of sorting gate. To examine the yield of sex-sorted sperm, X-bearing sex-sorted sperm from 4 Holstein bulls were produced repeatedly 34 times by 3 flow cytometers. The sizes of sorting gates were fixed at 40–42%, 44%, and 46%. Each yield of sorting gate at 40–42%, at 44% and at 46% was compared. To evaluate factors that affect purity, X- or Y-bearing sex-sorted sperm were produced by one flow cytometer. These trials were repeated 160 times for the sorting of X-bearing sperm and 45 times for the sorting of Y-bearing sperm. Stepwise multiple regression analysis was used to analyse the relationships between the purity of sex-sorted sperm and the following sorting conditions, the percentage of oriented sperm, the percentage of dead sperm, degree of separation between X-bearing and Y-bearing sperm, the size of sorting gate, event rate, drop drive frequency, drop delay value and drop delay accuracy. The highest yield was acquired by sorting gate at 44%. The number of sex-sorted sperm was increased as sorting gate was expanded, however, the purity became low. The purities of the sperm by some trials using sorting gate at 46% were less than our acceptable lowest purity that was 90%. So that those sperm must be discarded. Therefore the yield of sorting gate at 44% was greater than sorting gate at 46%. Stepwise multiple regression analysis revealed that the factors for increasing purity of X-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.001), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.001), the drop delay accuracy (P < 0.001), the event rate and the drop drive frequency, and the factor for decreasing purity of X-bearing sex-sorted sperm was the size of sorting gate (P < 0.001). On the other hand, the factors for increasing purity of Y-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.01), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.01) and the event rate (P < 0.05), and the factor for decreasing purity of Y-bearing sex-sorted sperm was the size of sorting gate (P < 0.01). From these results, it can be concluded that the purity of sex-sorted sperm was not depend on simply by size of sorting gate but was more completely explained by other sorting conditions.

85 DOMESTIC CAT KITTENS BORN AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERM

For successful application of sperm sorting technology to a particular species, the various assisted reproductive techniques required for in vivo or in vitro embryo production must be established. Previously, we have demonstrated the in vivo developmental competence of cat embryos derived by IVF 1) with presumptive X-chromosome bearing sperm and 2) after controlled-rate cryopreservation. Here, our intent was to determine in vivo viability by transfer to recipients of cryopreserved cat embryos produced by IVF with flow-sorted sperm. Semen collected from a single male using an artificial vagina was extended in electrolyte-free solution and shipped overnight (4°C) to the sorting facility. After sorting immediately upon arrival, sperm were concentrated by centrifugation, suspended in TEST yolk buffer, and return-shipped to the IVF laboratory at 4°C. At 48 h after semen collection, in vivo matured oocytes (n = 64) recovered from 2 gonadotropin-treated donors were inseminated in vitro with presumptive X-chromosome-bearing sperm. At 5 h post-insemination, oocytes were rinsed and placed in modified Tyrodes+BSA and NEAA (IVC-1) at 38°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. At 27 h post-insemination (Day 1), 43 two-cell embryos (67% cleavage rate) were equilibrated (3 steps) in 1.4 M propylene glycol, 0.125 M sucrose, and 10% dextran 70, cooled at a slow controlled rate (0.3°C min–1) from –6°C to –30°C, and plunged into liquid nitrogen. After 26 to 43 months in cryostorage, 4 straws, each containing 8 or 9 embryos, were thawed in air (22°C) and cryoprotectant was removed from the embryos by a 5-step rinse. Then, embryos were cultured for 17 to 20 h in IVC-1, as described earlier. On Day 2, 35 embryos were transferred (8 or 9 per recipient) by laparoscopy to the oviduct of four Day 1 gonadotropin-treated recipients at 24 h after laparoscopic oocyte retrieval (16 to 25 oocytes/donor). Most embryos (83%) were at the four- to eight-cell stages at transfer. Three recipients established pregnancies and delivered, without assistance, litters of 1, 2, and 3 live kittens (27% embryo survival) between Day 64 and Day 67 of gestation. Four kittens were female, and two were male. Birth weights ranged from 73 to 133g (mean = 117 g). Overall, of 18 kittens [12 from fresh (2009 Theriogenology, 71, 864) +6 from frozen embryos] born after transfer of embryos produced by IVF with sex-sorted sperm, 16 (89%) have been female. The variation in sex ratios between kittens born from fresh v. cryopreserved embryos probably is a reflection of the sort purity of the different sperm samples used for IVF. The ability to produce kittens after transfer of cryopreserved embryos derived by in vitro insemination with sex-sorted sperm represents an expansion in the assortment of assisted reproductive techniques that may be useful for selective propagation of biomedically relevant and endangered felids.

277 EFFECTS OF SEMINAL PLASMA ON SEX-SORTING BOVINE SPERM

This experiment evaluated how characteristics of bovine ejaculates affect the sortability of X- and Y-bearing spermatozoa. Ejaculates were collected by artificial vagina, 2 each from 10 bulls with an average of 1 h between collections. Only ejaculates with at least 60% motile and 70% normal sperm were used. Semen was centrifuged at 1 000 × g for 15 min to separate sperm from seminal plasma; seminal plasma was clarified by 10 min of additional centrifugation at 2 000 × g. Sperm were rediluted to 160 million sperm/mL with TALP (pH 7.4) and 0, 5, 10, or 20% seminal plasma, from the same ejaculate or reciprocally from first/second ejaculates. Following incubation with Hoechst 33342 for 45 min, an equal volume of TALP (pH 5.5) containing red food dye was added, and sperm were analysed on a MoFlo (Dako, Glostrup, Denmark) flow cytometer for percentage live-oriented cells, X sort rate, coincidence rate, percentage dead or dying (sperm membrane permeable to red dye), and splitability (peaks to valley ratio). The percentage live-oriented sperm was higher for treatments with 0% seminal plasma (64.4%) than for 5 (59.6%), 10 (59.0%), and 20% (57.8%) seminal plasma (P < 0.01). The percentage live-oriented sperm was higher for second (63.0%) than for first ejaculates (56.2%). Sort rate was higher for 0% seminal plasma and second ejaculates (P < 0.05). Dead/dying rates were lower for 0% (16.5%) than for 5 (21.9%), 10 (23.6%), or 20% (23.4%) seminal plasma (P < 0.003); and for first ejaculates (25.9%) compared with second ejaculates (18.2%). Seminal plasma percentage and ejaculate had no effect on splitability, and there was no difference in sorting parameters whether the seminal plasma was from the first or second ejaculate. The initial sperm concentration of the ejaculate (range, 1.3 to 3.4 billion sperm/mL) did not affect any sperm sorting parameter. Thus, effects of sperm concentration on sortability need to be reconsidered, as current practice is to select ejaculates to sort based on initial sperm concentration. The initial pH, percentage morphological abnormalities, initial seminal plasma pH, and age of bull did not affect sorting parameters. In conclusion, the presence of seminal plasma during staining and sorting may decrease sort rate and percentage of live-oriented cells, as well as increase death rate. In addition, sorting second ejaculates may be more advantageous than sorting first ejaculates. Future studies are needed to determine if the results hold true if sperm are stored for several hours and how the various factors affect sperm post-thaw viability.

A Noninvasive, Motility Independent, Sperm Sorting Method and Technology to Identify and Retrieve Individual Viable Nonmotile Sperm for Intracytoplasmic Sperm Injection

For intracytoplasmic sperm injection in the absence of sperm motility it can be virtually impossible to distinguish viable from nonviable sperm. A reliable means to identify viable nonmotile sperm is needed and would likely improve the intracytoplasmic sperm injection success rate. Optoelectronic tweezers are a new technology that uses light induced dielectrophoresis fields to distinguish individual live cells from dead cells. We assessed the ability of optoelectronic tweezers to distinguish viable from nonviable individual nonmotile human sperm. Fresh semen specimens from 6 healthy men were suspended in an isotonic sucrose/dextrose solution and incubated with 0.4% trypan blue dye (Sigma-Aldrich®). Within 15 minutes we randomly selected 5 motile and 50 nonmotile sperm, including 25 trypan negative, followed by 25 trypan positive sperm, under 200× magnification for optoelectronic tweezers assay. We recorded the individual sperm response (attraction or repulsion) to the optoelectronic tweezer field and trypan staining status. From each subject 55 unwashed sperm were individually assayed for a total of 330. All motile sperm were attracted to optoelectronic tweezers. Of 150 trypan negative (viable) sperm 132 (88%) were attracted to the optoelectronic tweezer field with 0.88 sensitivity (95% CI 0.82-0.93) vs that of the trypan blue assay. All 150 trypan positive (nonviable) sperm were repulsed by or neutral to the optoelectronic tweezer field with 1.0 specificity (95% CI 0.98-1.00) vs that of the trypan blue assay. Type I error equaled 0 and overall assay agreement was 94%. The optoelectronic tweezer assay can distinguish viable from nonviable nonmotile viable sperm with sensitivity comparable to that of the trypan blue assay and equal specificity. Optoelectronic tweezers are a promising means of selecting sperm for intracytoplasmic sperm injection.

Effects of antioxidants on boar spermatozoa during sorting and storage

Sorting procedures submit sperm cells to a set of stressful steps that can trigger an increase of ROS production and consequently reduce sorted semen quality. This study evaluated the effect of supplementation with different antioxidants (EGCG, Na pyruvate + catalase, SOD) on acrosome and plasma membrane integrity, activation of caspases (as assayed by FITC-VAD/PI staining) and immunolocalization of Hsp70 of boar spermatozoa after sperm preparation (Hoechst 33342 staining) and sorting procedure. The effect of antioxidants, with or without seminal plasma, on sorted spermatozoa stored for 24 h at 15 °C was also evaluated. Antioxidants did not exert any preventive action on sperm modification induced by staining and sorting. After 24 h of storage at 15 °C, sorted samples supplemented with either EGCG or SOD plus seminal plasma showed a significant ( p < 0.05) increase of the percentage of viable spermatozoa, while no positive effect on the other tested parameters was observed; EGCG seems to exert an inhibition on caspase activation in that a decrease of the number of dead cells FITC-VAD+/PI+ was recorded. In conclusion, our results indicate that EGCG and SOD in association with seminal plasma are effective in exerting some compensatory protection against the detrimental effects of storage of sorted semen while their action is not evident during steps of the sorting procedure.

The Ethics of Sex Selection and Family Balancing

Ethical concerns about the ethics of selecting the sex of a child predate current techniques of prenatal genetic diagnosis (PGD) and sperm sorting. The only methods previously available were highly problematic, as they involved infanticide or abortion of an unwanted sex. PGD is less problematic than the earlier methods, yet still troubling to some because it involves destruction of a healthy embryo and risks to women. The technique of sperm sorting, still in an experimental phase, is the least ethically problematic method, yet opponents argue that sex selection by any means involves sex discrimination and can have undesirable consequences. One such consequence is an imbalance in the sex ratio. This imbalance already exists in some Asian countries that favor male children, but is less likely in Western Europe and North America. There is increasing acceptance of family balancing as a reason for sex selection, but some people remain opposed to broadening the indications for sex selection of offspring beyond family balancing. Nevertheless, parents may have good reasons other than family balancing for choosing the sex of a future child. Such reasons may be justified by the principle of reproductive liberty.

Should Human Beings Have Sex? Sexual Dimorphism and Human Enhancement

Since the first sex reassignment operations were performed, individual sex has come to be, to some extent at least, a technological artifact. The existence of sperm sorting technology, and of prenatal determination of fetal sex via ultrasound along with the option of termination, means that we now have the power to choose the sex of our children. An influential contemporary line of thought about medical ethics suggests that we should use technology to serve the welfare of individuals and to remove limitations on the opportunities available to them. I argue that, if these are our goals, we may do well to move towards a “post sex” humanity. Until we have the technology to produce genuine hermaphrodites, the most efficient way to do this is to use sex selection technology to ensure that only girl children are born. There are significant restrictions on the opportunities available to men, around gestation, childbirth, and breast-feeding, which will be extremely difficult to overcome via social or technological mechanisms for the foreseeable future. Women also have longer life expectancies than men. Girl babies therefore have a significantly more “open” future than boy babies. Resisting the conclusion that we should ensure that all children are born the same sex will require insisting that sexual difference is natural to human beings and that we should not use technology to reshape humanity beyond certain natural limits. The real concern of my paper, then, is the moral significance of the idea of a normal human body in modern medicine.

Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis

Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10 8 cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10 4 to 575.0 ± 56.3 × 10 4 cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

Probes and Techniques for Sperm Evaluation by Flow Cytometry

Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non-sperm particles in the samples ('debris'). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered.

Single and twin ongoing pregnancies in two cases of previous ART failure after ICSI performed with sperm sorted using annexin V microbeads

To treat couples with intracytoplasmic sperm injection (ICSI) after annexin V sperm sorting. Two case reports. Department of Reproductive Medicine at a private medical institute. Couples on infertility treatment, donor oocytes. Sperm sorted with annexin V magnetic microbeads before ICSI, day 3 embryo transfer; case 1: ovum donation; case 2: patient oocytes. 1) Sperm DNA fragmentation (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling [TUNEL]) and active caspase-3 (immunocytochemistry); 2) fertilization rate, embryonic quality, blastocyst development of nontransferred embryos, and pregnancy outcome after ICSI of sorted sperm. Case 1: Premature ovarian failure patient with previous fertilization failures: asthenoteratozoospermia, abnormal DNA fragmentation (TUNEL 30%; normal <20%). ICSI with annexin V-treated sperm done on six donated metaphase II (MII) oocytes; four fertilized, and a 5-cell/grade-2 and a 6-cell/grade-2-3 embryo were transfered. A day 5 blastocyst was cryopreserved. The patient was in the last trimester of gestation. Case 2: Couple with >4 years of primary infertility and recent ICSI failure. Semen with teratozoospermia (5% normal forms [Kruger]) and abnormal active caspase-3 (16%; normal <11%). ICSI with annexin V-treated sperm done on 9 MII oocytes. All fertilized; a 7-cell/grade-1 and an 8-cell/grade-1-2 embryo were transferred. A day 5 expanded blastocyst was cryopreserved. The patient was in the second trimester of a twin normal pregnancy. Sperm sorting with annexin V columns was effective in the treatment of two cases of ICSI failure, resulting in a single and a twin pregnancy after transfer of two embryos in each case.