Sperm sexing in sheep and cattle: the exception and the rule

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.     

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.     

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.     

Field fertility of sex-sorted and non-sorted frozen-thawed stallion spermatozoa

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen–thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197–201] was used to deposit low doses (6, 13 or 25 × 10⁶ frozen–thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 × 10⁶ spermatozoa, 500 μL) obtained after artificial insemination with frozen SS spermatozoa (n = 29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 × 10⁶ NS spermatozoa in 250 μL (n = 31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 × 10⁶ motile NS frozen–thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen–thawed rather than sex-sorted spermatozoa.     

Sex preselection of sٍheep embryo by altering the minerals of maternal nutrition

Several methods have been conducted for embryo sex preselection, which includes X- and Y- sperm separation, changing the pH of the female reproductive tract, time of mating before or after ovulation, and feeding formula, such as altering the presence of minerals in diet content before breeding may affect the embryo sex preselection ratio. In this study, three food formulas to feed female sheep were created with the cooperation of the Arabian Agricultural Services Company (Arasco). Ewes were fed with modified food formulas for one month before mating with males. The first group (A) (30 ewes), modified for male embryo gender preselection, were fed a diet with an increased percentage of the minerals Na⁺, K^+, and P^-. The second group (B) (30 ewes), modified for female sex preselection, were fed a diet with an increased percentage of the minerals Ca⁺⁺ and Mg⁺⁺. The third (control) group (C) (30 ewes) were fed the regular (Wafi) food formula. Our results showed no significant differences were in mean body weights between the three groups at the end of the feeding period. The results of different feeding formulas on mineral serum blood samples of ewes showed an increase in Na⁺, K^+, and Cl^- ions in the serum of group (A) compared to the other groups (B and C). The concentration of Na⁺ in the serum of group (A) was significantly (P < 0.05) higher than group (C). The concentration of Cl^- ions in serum samples of ewes in group (A) was significantly higher than group (C) and group (B) (P < 0.05). The role of maternal feeding on embryo sex preselection shows that the pregnancy rate of animals in group (A) was 73.33%. Group (A) birthed 17 males and 5 females (77.27% and 22.72%, respectively). The pregnancy rate in group (B) was 70%. Group (B) birthed 6 males (27.27%) and 16 females (72.72%). Finally, the control group (C) had a pregnancy rate of 76.66%. They birthed 13 males (54.41%) and 11 females (44.83%). The results of our study confirm that altering the percentage of minerals in the maternal diet plays a role in sex preselection in sheep, which agree with other mammalian studies in rats and mice. Thus, the result of this study can help farmers to manage their breeding. We recommend that more studies on the relationship between minerals in the diet should be conducted for other spices and human sex preselection.     

Advances in flow cytometry for sperm sexing

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s⁻¹ for an input rate of 40,000 X- and Y- sperm s⁻¹. With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 × 10⁶ sperm. The sort rate of 8000 sperm s⁻¹ mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

Pan-European phylogeography of the aquatic snail Theodoxus fluviatilis (Gastropoda: Neritidae)

Investigating the geographical distribution of genetic lineages within species is critical to our understanding of how species evolve. As many species inhabit large and complex ranges, it is important that phylogeographical research take into account the entire range of widespread species to clarify how myriad extrinsic variables have affected their evolutionary history. Using phylogenetic, nested clade, and mismatch distribution analyses on a portion of the mitochondrial COI gene, I demonstrate that the wide-ranging freshwater snail Theodoxus fluviatilis possesses in parallel many of the phylogeographical patterns seen in less widespread freshwater species of Europe. Fragmentary forces play a major part in structuring the range of this species, with 12 of 14 geographically structured nested clades displaying a distribution consistent with fragmentation or restricted dispersal. Certain regions of southern Europe harbour the majority of genetic diversity (total haplotype diversity, H = 0.87), particularly Italy (H = 0.87) and areas surrounding the Black Sea (H = 0.81). Post-Pleistocene range expansion is pronounced, with the majority of northern European populations (95% of sample sites) having arisen from northern Italian individuals that initially colonized northern Germany. Additionally, two highly divergent haplotype lineages present in northern Germany imply that there were at least two postglacial recolonization routes. Estuaries may also provide a means of dispersal given that no genetic differentiation was found between estuarine populations and neighbouring freshwater populations. Taken together, these data reveal a species with a complex genetic history resulting from the fragmentary effects of European geology as well as continuous and discrete range expansion related to their aquatic biology.     

Considering evolutionary processes in the use of single-locus genetic data for conservation,with examples from the Lepidoptera

The increasing popularity of molecular taxonomy will undoubtedly have a major impact on the practice of conservation biology. The appeal of such approaches is undeniable since they will clearly be an asset in rapid biological assessments of poorly known taxa or unexplored areas, and for discovery of cryptic biodiversity. However, as an approach for diagnosing units for conservation, some caution is warranted. The essential issue is that mitochondrial DNA variation is unlikely to be causally related to, and thus correlated with, ecologically important components of fitness. This is true for DNA barcoding, molecular taxonomy in general, or any technique that relies on variation at a single, presumed neutral locus. Given that natural selection operates on a time scale that is often much more rapid than the rates of mutation and allele frequency changes due to genetic drift, neutral genetic variation at a single locus can be a poor predictor of adaptive variation within or among species. Furthermore, reticulate processes, such as introgressive hybridization, may also constrain the utility of molecular taxonomy to accurately detect significant units for conservation. A survey of published genetic data from the Lepidoptera indicates that these problems may be more prevalent than previously suspected. Molecular approaches must be used with caution for conservation genetics which is best accomplished using large sample sizes over extensive geography in addition to data from multiple loci. Matthew L. Forister, Chris C. Nice and James A. Fordyce contributed equally to this paper.     

One species or two? Multilocus analysis of nucleotide variation of Melastoma penicillatum and Melastoma sanguineum (Melastomataceae) in Hainan,China

Melastoma comprises more than 20 shrub species and is distributed in tropical Asia and Oceania. Melastoma penicillatum, the most narrowly distributed taxon of this genus in China, was recently incorporated into another morphologically similar species, M. sanguineum. Based on its distinct morphological traits, unique habitat and flowering time, we propose that M. penicillatum should be a distinct species. In this study, we sequenced three nuclear genes and four chloroplast intergenic spacers of M. sanguineum and M. penicillatum at two locations in Hainan to test this hypothesis. There was no sequence variation at all four chloroplast intergenic spacers within and between M. sanguineum and M. penicillatum, suggesting that they should have diverged recently. However, they showed strong divergence at all three nuclear genes and no shared haplotypes were observed between them. Bayesian clustering-based STRUCTURE analysis revealed that individuals of M. sanguineum and M. penicillatum were clustered into their own clades. Fst calculations showed that both taxa had marked population genetic differentiation, but population differentiation in M. penicillatum was stronger than that in M. sanguineum. Stronger population differentiation in M. penicillatum may be caused by its discontinuous high-elevation and understory habitats, which is disadvantageous for bird-mediated dispersal. Our analysis based on the isolation-with-migration (IM) model revealed bidirectional, asymmetrical gene flow in M. sanguineum and little gene flow in M. penicillatum. Taken together, our results indicate that M. penicillatum is a distinct species and should not be incorporated into M. sanguineum. The two species might have diverged recently, and lineage sorting has been incomplete. Differential adaptation to different elevations might have led to speciation in this case.     

Analysis of cellular DNA distributions

In the fluorescent-flow cytophotometric measurement of cellular DNA content the DNA distributions usually have two peaks. The second peak, which corresponds to the 4C DNA content of G2 and M cells, is often positioned at lower values of DNA content than twice that of the 2C DNA peak which contains G1 cells. Computerized numerical analyses were performed on artificial DNA distributions in which the proportion of S-phase cells was varied. It was demonstrated that the contribution of late S-phase cells to the 4C DNA peak in the histogram shifts the second peak to a position below twice the 2C DNA value. Also, increasing the coefficient of variation of the DNA measurement shifts the second peak position to lower values. A group of 33 DNA distribution histograms was found to have an average G2/G1 peak position ratio of 1.90, in keeping with typical values obtained from the numerical analysis of the artificial populations.     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.     

DNA分析与基因组序列和植物系统学研究

从DNA杂交、RFLP分析、DNA的限制酶图谱分析等方面描述DNA分析技术在植物学研究中的应用,讨论了DNA分析技术与植物系统学的关系及分子数据的分析方法。并以高等植物为对象,从核DNA、叶绿体DNA和线粒体DNA三方面对植物分子系统学进行了论述。