Unsorted Sperm Research Articles

Fertilization potential test of sperm from nano monoclonal antibody injected goats

• Over 90 % of X bearing sperm could be available using NMA™ sex selection technique. • In vitro fertility potential of sperm sorted by NMA™ injection was comparable to that in the control group. • The percentage of female offspring was more than 90 %. Controlling the sex of an animal’s offspring has long been the dream of most livestock producers. To produce a very higher percentage of X chromosome bearing sperm, calcium phosphate nano monoclonal antibody (NMA™) against Guanzhong dairy goat sperm epitope was developed in advance, and the detailed development technique has been applied for patent protection. The present study focused on the fertilization potential detection of sperm which was treated by testis injection of NMA™ to implementing sex selection of Guanzhong dairy goat. Results showed that no significant differences in sperm viability, sperm velocities, acrosomal integrity and plasma membrane integrity, although the volume and density of a single ejaculate were slightly lower in goats from the NMA™ injection group than those of the control group. From the 6th to 30th week following NMA™ injection, the percentage of X chromosome bearing sperm was more than 90 %; at the week 40th, it dropped to 88 %. After the second NMA™ injection performed at the 40th week, over 90 % of sperm with X chromosome could be obtained again. Moreover, the in vitro fertilization capacity of sperm derived from NMA™ injection was the same as that of the control group. No significant difference in pregnancy rates and kidding rates was observed following artificial insemination using sorted sperm versus unsorted sperm, but the proportion of female offspring was more than 90 %, which was significantly higher in the sex sorted group than that of the control group. The sex determination technique presented here can be mainly applied to facilitate the mass production of female Guanzhong dairy goats.

P–121 Selection and separation of X- and Y- chromosome-bearing mammalian sperm using SPERMAN technology

Abstract Study question How can we select and separate X and Y-chromosome bearing sperm cells without causing cellular damage and reduced fertility? Summary answer AI powered SPERMAN technology can sort X and Y-chromosome bearing sperm populations without using harmful fluorescent dyes and lasers. What is known already Most common and reliable method to sex sorting (separation of X and Y- chromosome bearing sperm cells) is by using fluorescence activated cell sorting ( FACS) which takes advantage of the difference of amount of DNA in X and Y-chromosomes. Unfortunately this method causes reduced fertility and cellular damages due to the lasers and fluorescent dyes that are used. There are new and experimental developments in sex-sorting such as using immunological approaches to separate sperm cells. Current sorting method damages sperm cells. However, there is no automated and easy to use sperm sorting technology available. Study design, size, duration In this study, we have compared the quality parameters (motility, viability, AR status, DNA packaging) of sorted sperm cells to unsorted sperm cells. We have used epididymal sperm samples from 10 C57BL/6 mice (Charles River) and frozen semen samples from 6 Holstein bulls (Sexing Technologies) and frozen semen samples from 8 human sperm donors (Fairfax Cryobank). Samples were divided into two groups: sex-sorted and unsorted sperm. Participants/materials, setting, methods Sperm samples from 8-week-old C57BL/6 mice were collected from epididymal region, frozen bull semen samples and human semen samples were thawed at 37ºC then separated into two groups as sorted and unsorted sperm. Sorted samples in modified SP-TALP medium were loaded on the microfluidic chip of Sperman device, after the 30min long sorting process ended, all samples were centrifuged gently at 100g for 5 min, quality parameters were measured by using CASA and FACS. Main results and the role of chance Sperman device sorted X-bearing sperm cells at 81% purity and Y-bearing cells at 73% in mouse samples. Sperman device was able to sort X-bearing sperm cells at 78% purity and Y-bearing cells at 70% in bull samples.S perman device sorted X-bearing sperm cells at 85% purity and Y-bearing cells at 76% in human samples. Our study shows that in mouse, bull and human sperm samples, sperm DNA quality, sperm concentration, progressive motility and AR status results from the sorting with SPERMAN device are comparable with the unsorted samples. For sperm DNA quality, both the Spearman rank correlation coefficient and concordance correlation coefficient are above 0.97, indicating a high agreement between the unsorted samples and SPERMAN sorted samples. Limitations, reasons for caution In this study we only compared sperm quality parameters between unsorted sperm cells and Sperman-sorted sperm cells. Ideally a follow up study would be to look in detail at fertilization success rates and embryo growth. Also, genomic and proteomic profiles of unsorted and sorted sperm cells could be different. Wider implications of the findings: In livestock, sperm sex sorting has high value in terms of economic impact on the livestock management. Unfortunately, there is one expensive single technology dominates the sex-sorting market causing high fees poor outcomes. We believe that Sperman technology provides farmers more cheaper and better sex-sorting technology. Trial registration number Not applicable

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p<.05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p<.05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system.

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows: Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p<.05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p<.05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.

Enrichment of bovine X-sperm using microfluidic dielectrophoretic chip: A proof-of- concept study

The microfluidic dielectrophoretic (MF-DEP) chip is a new, economical and readily-available technology that might be used to enrich X-sperm for increasing female offspring in dairy farms. In this study, we sought to develop an MF-DEP chip to enrich X bovine sperm. The MF-DEP chip was composed of an electrode attached to a glass slide and a microchannel made from polydimethylsiloxane. Sex-sorted sperm from flow cytometry were used to identify optimal electric field conditions while unsorted sperm were later tested for sorting efficiency. The results show that during dielectrophoresis some sperm attached to the electrode (called positive DEP; pDEP) whereas other moved away from the electrode (called negative DEP; nDEP). X and Y-sperm responded to dielectrophoresis differently depending on various factors such as buffers, voltages, and frequencies. We found that the condition 4 V 1 MHz significantly reduced (P < 0.05) the percentage of Y-sperm to nearly 30 and therefore enriched X-sperm. The sorting efficiency was dependent on buffer, bull, sorting cycle, flow rate, electrical voltage, and frequency. Notably, the best sorting buffer found in this experiment was the conducting buffer, but this buffer significantly reduced sperm viability and motility. Other sperm-friendly buffers, TRIS and mHTF, were also used, but could not enrich X-sperm. In conclusion, this is a proof of concept that the MF-DEP chip can be effectively used to enrich bovine X-sperm. However, more research must be performed particularly to find the best sorting buffer to effectively sex-sort sperm while providing high motility and sperm viability.

The bovine sex-determining region Y (Sry) gene and its mRNA transcript are present in Y sperm but not X sperm of bulls

AbstractThe aims of this study were to establish whether the sex-determining region Y gene and its mRNA transcript are present in the Y sperm and X sperm of bulls and, if present, determine their cellular localization. Semen was collected from three bulls and sorted by flow cytometry into X- and Y-chromosome populations. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determineSrymRNA expression in X sperm and Y sperm. The presence and localization ofSryDNA and RNA were investigated by fluorescence in situ hybridization (FISH). RT-PCR detected a singleSrytranscript of 142 bp in Y sperm but not in X sperm. In Y sperm, the FISH-positive rates forSryDNA andSryRNA did not differ significantly from the re-analyzed Y sperm purity. In further experiments, there were no significant differences between the FISH-positive rate forSryRNA and the re-analyzed Y sperm purity for X-sorted, Y-sorted, or unsorted sperm. In conclusion, FISH analysis revealed thatSrytranscripts are present at the edges of the sperm heads of Y sperm but are absent from X sperm.

Deep insemination with sex-sorted Cashmere goat sperm processed in the presence of antioxidants.

Flow cytometrically sex-sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex-sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex-sorting and freezing process, including ascorbic acid-2-glucoside AA-2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex-sorted fresh and frozen-thawed sperm. Based on these experiments, we performed deep insemination with sex-sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA-2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA-2G supplementation showed an evident protection against the sorting process-induced membrane and acrosome damage. In Experiment 2, AA-2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen-thawed sex-sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA-2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA-2G supplementation efficiently protects quality and function of both fresh and frozen-thawed sex-sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.

The quality of sexing-sorted bull sperm after in vitro capacitation

Kaiin EM, Gunawan M. 2017. The quality of sexing-sorted bull sperm after in vitro capacitation. Pros Sem Nas Masy Biodiv Indon 3: 466-470. Sexing-sorted bull sperm separated with three column BSA (5-10%) was evaluated in vitro to determine the quality of sperm after capacitation. The frozen FH bull sexing-sorted X (female) and Y (male) sperms were re-thawed at water bath with a temperature of 37oC for 30 seconds. Then, the quality of sperms was evaluated in the parameters of motility, viability and abnormality. The state of sperm capacity was examined with triple staining at each sample, before and after capacitation. X and Y sperms were capacitated in two treatment media, i.e., TALP medium with 4 mM caffein (I), or 4 mM caffein combined with heparin 10 µg/ml (II). Unsorted sperm was used as a control. The research was conducted in five repetitions, by using X, Y, and unsorted sperms. The results showed that the motility of X dan Y sperms at the capacitation medium with caffein and heparin was significantly higher (X1=50.9%, X2=48.6%, and Y=51.5%) compared to the capacitation medium only with caffein (X1=41.5%; X2=43.9% and Y=41.3%). The same result was obtained from unsexed sperm. The viability of X dan Y sperm capacitated with medium II (X1=66.9%, X2=61,7%, and Y=60.3%), also resulted in a higher percentage compared to medium I (X1=54.8%, X2=50.6%, and Y=54.9%). The abnormality of X and Y sperms after capacitation at both treatment medium was not different significantly compared to unsorted sperm. Before capacitation, the percentages of X and Y sperms with acrosome reaction were lower (2.6% and 0.5%), but after capacitation, it resulted in increasing the number of sperms with acrosome reaction incapacitation, both on medium I and II. This result showed that sexing-sorted bull sperm with BSA column not resulting in the earlier acrosome reaction before capacitation.

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Significant heparin effect on bovine embryo development during sexed in vitro fertilization.

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

Sperm pretreatment with glutathione improves IVF embryos development through increasing the viability and antioxidative capacity of sex-sorted and unsorted bull semen

The antioxidant of reduced glutathione (GSH) is the most abundant thiol in cells for the maintenance of the intracellular redox balance. The study aimed to assay the effect of sperm treatment with GSH before incubation with oocytes on the development potential of embryos obtained by in vitro fertilization (IVF). Also the mitochondrial membrane potential (ΔΦm), plasma membrane integrity (viability), DNA fragmentation, reactive oxygen species (ROS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, methane dicarboxylic aldehyde (MDA) level as indices of lipid peroxidation in sex-sorted and unsorted sperm from three bulls were investigated using flow cytometry and enzyme-labeled instrument individually. The results showed that 2 mmol L−1 GSH increased significantly the cleavage rate (86.68% vs. 82.78%), 4- to 8-cell rate (82.30% vs. 73.43%) and blastocyst rate (43.15% vs. 35.24%) of IVF embryos compared with untreated group. Furthermore, addition of GSH increased significantly the ΔΦm and viability, decreased the ratio of DNA fragmentation in sex-sorted or unsorted semen (P<0.05), except the sex-sorted semen from bull 019. Similarly, activities of SOD, CAT and GPx were increased significantly. However, the contents of MDA were decreased significantly both in sex-sorted and unsorted semen treated with GSH (P<0.05). These results suggest that sperm pretreatment with GSH during IVF can maintain better the viability and fertility of sperm through reducing apoptosis and increasing the antioxidant capacity, which improves the IVF embryos development.

173 IN VITRO EMBRYO PRODUCTION FROM OOCYTES FERTILISED WITH UNSORTED OR X-SORTED SPERM AND ISSUED FROM SUBFERTILE HIGH GENETIC MERIT COWS SUBMITTED OR NOT TO A 48-h COASTING PERIOD FOLLOWING FSH STIMULATION

High genetic merit cows may be sidelined from breeding schemes because of reproductive disorders. In vitro production of embryos (IVP) issued from ovum pickup (OPU) can be an alternative to bypass infertility problems as experienced in humans and thus accelerate genetic progress (Duszewska et al. 2012 J. Anim. Feed Sci. 21, 217–233). The aim of this work was to evaluate if IVP from high genetic merit subfertile cows could benefit to the breeding scheme under commercial conditions, at the Biotechnology MIDATEST Station located in Denguin, Southwest, France. Holstein cows (n = 16) from 3.5 to 13 years old with different reproductive pathologic problems (repeated breeding, failure in in vivo embryo production, embryo mortality, permanent cysts, oviduct infection) were used in an OPU-IVP program. Donor cows were stimulated with decreasing doses pFSH twice daily during 3 days (Stimufol®, total dose: 350 µg of pFSH). The OPU was performed 48 h after the last FSH injection in the “coasting” group v. 12 h after the last FSH injection for “no-coasting” group and in vitro matured using a standard IVM protocol. Oocytes were fertilised with frozen-thawed unsorted or X-sorted sperm in TALP medium from different bulls (n = 42) without any previous IVP testing. Presumptive zygotes were cultured in SOF medium (Minitub®) plus 1% cow serum up to Day 7 at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. The OPU-IVP was repeated 1 to 6 times (3.75 ± 2.6) for each donor cow. Grade 1 blastocysts and expanded blastocysts (according to IETS classification) were recorded on Days 6.5 and 7. Embryo production was analysed with ANOVA and blastocyst yield was analysed by chi-square. The results in both coasting or no-coasting groups and the effect of fertilization using unsorted or X-sorted sperm are presented in Table 1. The embryonic development rate was significantly higher when using unsorted semen to fertilize the oocytes compared to X-sorted sperm (P &lt; 0.05). On the other hand the coasting period had no significant effect neither on the number of collected oocytes nor on the embryonic development rates. In conclusion, our work confirmed the efficacy of OPU-IVP techniques to produce grade 1 embryos using X-sorted or unsorted sperm in subfertile high genetic merit cows. Table 1.Oocyte collection and in vitro embryo production, in donor cows submitted or not to a 48-h coasting period and effect of fertilization with unsorted v. X-sorted sperm

Influence of Sex on Basal and Dickkopf-1 Regulated Gene Expression in the Bovine Morula.

Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.

137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P &lt; 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).

Tentative identification of sex-specific antibodies and their application for screening bovine sperm proteins for sex-specificity

Our previous studies indicated that a purified rabbit antiserum against X-sperm contained sex-specific antibodies (SSAbs) which preferentially bound to sex-sorted X-sperm. The specificity of sex-specific antiserum was initially demonstrated using flow cytometry only, which resulted in uncertainty. In this study, the putative SSAbs against bovine X-sperm (XSSAb) were produced by a series of immunological approaches, and the effectiveness of separation of sperm using putative XSSAb was validated. Subsequently, the XSSAb was used to immunoprecipitate sex-specific proteins (SSPs) in bovine sperm, followed by two-dimensional gel electrophoresis. The results showed 7.6, 15.2 and 52.1 % of sex-sorted Y-sperm, sex-sorted X-sperm and unsorted sperm were recognized by the neutralized rabbit antisera against X-sperm, respectively. Also the purity of separation of sperm using putative XSSAb reached 74.3 % when the immunologically separated sperm were injected into oocytes. In addition, three candidate SSP sports about 30 kDa were captured by the XSSAb. Our results confirmed that the putative XSSAb contained SSAbs, and implied that these three protein sports might be SSPs in bovine X-sperm. This provides a potentially more efficient method for sorting sperm and lays a foundation for future search for SSPs.

Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions

The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 106 unsorted sperm per oviduct; (2) single LI with 0.5 × 106 sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 106 sex-sorted sperm per oviduct and 0.5 × 106 sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 106 sperm per oviduct and 1 × 106 sperm per uterine horn; or (2) 1 × 106 sperm per oviduct and 2 × 106 sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 106 sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.

Use of sex-sorted and unsorted frozen/thawed sperm and in vitro fertilization events in bovine oocytes derived from ultrasound-guided aspiration

The objective was to investigate the effects of sex-sorting on early fertilization events in ovum pick up (OPU) derived oocytes fertilized in vitro with frozen-thawed sperm at different co-incubation lengths. Eighty-four OPU sessions were carried out in 18 cyclic, dry and non-stimulated Holstein Friesian and German black pied cows. Ovum pick up oocytes were matured in vitro for 24 hours and fertilized with frozen-thawed sex sorted or unsorted sperm from the same ejaculate. Fertilization was achieved by two experimental protocols: 1) short gamete co-incubation length: 4, 8 and 12 hours; and 2) long gamete co-incubation length: 18 and 24 hours. After in vitro fertilization, ova were fixed and stained to identify early fertilization events. Sperm penetration, monospermy, pronuclear formation and syngamy did not differ, whether sexed or unsexed sperm was used. Overall, the findings demonstrate similar fertilizing potential between sex-sorted and unsorted sperm.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.

Seminal plasma and seminal plasma proteins added to bulk sorted sperm do not alter the mRNA expression of in vitro produced bovine embryos

Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL−1; n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO2, 5% N2). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1−4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.

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.