Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.     

Cryopreservation of flow-sorted bovine spermatozoa

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.     

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.     

Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA

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.     

What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?

Until now it has been unclear to what extent the reduced fertility with sexed semen in the dairy industry is caused by too few sperm per AI dose, or by the effect of flow cytometric sorting, which is the established procedure for sexing semen. Therefore, we evaluated the effects of low sperm numbers per dose with and without sorting on non-return rates after 56 days (NRR56); in addition, we evaluated the effects of bulls, in order to further optimize use of sexed semen.Based on results of using sexed semen from seven Holstein bulls, an overall numerical decline of 13.6% in NRR56 was observed (P < 0.05). About two-thirds of this decline (8.6%) was due to the low dose (P < 0.05), and a third (5.0%) due to the process of sorting (P < 0.05). The effect of low dosage and sorting differed among bulls. We observed a sex ratio of 91.6% females for sexed semen from the first 131 calves born.Currently the best way to increase fertility of sexed semen is by closely monitoring fertility so that the highest fertility bulls are used, and by improving farm animal management. However, to make substantial progress, more in depth studies are needed on the sexing technology, especially on aspects such as sorting procedures and sperm dosage.     

Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Application of sexed semen technology to in vitro embryo production in cattle

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. For thousands of years, livestock owners have desired a methodology to predetermine the sex of offspring for their herds. The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers' sex selection dreams reality in the 1990s and now semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter. Several concerns regarding the implementation of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. These issues are discussed in this review. There are also a number of issues that appear to influence the success rates of using sexed semen to produce bovine embryos in vitro. These issues include reductions in fertilization rates, lower cleavage rates, blastocyst rates and pregnancy rates, partial capacitation of the sperm, dilute sperm samples and sire variation. These subjects are also addressed in this paper. Finally, we will describe a recent field trial in which female Holstein embryos produced using the combined technologies of sex-selected semen and microfluidics were transferred either as single or bilateral twin embryos into beef cattle recipients, demonstrating these technologies' contributions to viable embryo production. The results indicate that large-scale transfer of in vitro produced, Holstein heifer embryos to beef recipients is a feasible production scheme.     

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

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

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm

Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n = 5) from one male were extended in electrolyte-free solution and shipped overnight at 4 °C to the sorting facility. Samples were adjusted to 75 × 10⁶ sperm/mL and stained with Hoechst 33342. After 1 h at 34.5 °C, samples were adjusted to 50 × 10⁶ sperm/mL with 4% egg yolk TALP + 0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P > 0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.     

Comparative motility of X and Y chromosome–bearing bovine sperm separated on the basis of DNA content by flow sorting

A combination of flow cytometric sperm sorting of X and Y chromosome–bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 × 10⁶ sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37°C) and their motion video recorded for 2 min using a magnification of ×100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR. Mol. Reprod. Dev. 50:323–327, 1998. Published 1998 Wiley-Liss, Inc.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm

Hoechst 33342 is the fluorophore used routinely to measure DNA in X- and Y-chromosome-bearing mammalian sperm so they can be separated by flow sorting. A difference of <3% in DNA mass can be detected. This synthetic dye consists of two adjacent benzimidazole rings with N-methyl-piperazine and phenolic groups at the ends. The molecule permeates the cell membrane of living cells and binds selectively to A-T base pairs exposed in the minor-groove of double stranded DNA. Capability to distinguish and separate X- and Y-chromosome-bearing sperm has led to artificial insemination of somewhere around a million female mammals. Offspring with obvious abnormalities are no more frequent than after insemination of unsorted sperm into cows, horses, humans, pigs, sheep, rabbits, dolphins and other mammals. There is no apparent genotoxic effect from exposure of sperm to Hoechst 33342, although information on cellular toxicity or development of embryos resulting from Hoechst 33342-stained sperm is less reassuring. Little is known about the fate of sperm-delivered Hoechst dye in the female reproductive tract or on progeny of resultant offspring.     

The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen‐thawed, unsorted, and sex‐sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non‐sexed (NS), sexed for X‐sperm (SX), and sexed for Y‐sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P = 0.09) or IGF2R genes (P = 0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex‐sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:77–84, 2012. © 2011 Wiley Periodicals, Inc.     

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

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.