Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.     

A method for chromosome analysis of ram spermatozoa using zona-free hamster oocytes

A method for displaying ram spermatozoan chromosomes using the interspecific zona-free hamster oocyte penetration was described to distinguish X- and Y-bearing spermatozoa. Semen samples from four rams were frozen and stored in liquid nitrogen. After thawing the samples, motile spermatozoa were collected by the swim-up method and treated with ionophore A23187 for the purpose of facilitating their capacitation. Slides were prepared by the gradual fixation-air dry method. The rates of oocyte penetration, first cleavage metaphase, and the number of ova that were karyotyped successfully were 67.9, 60.8 and 40.6%, respectively. The overall success rate (number of spermatozoa karyotyped/number of oocytes used for insemination) was 47.9%. A total of 1009 spermatozoa were analyzed, and the ratio of X- and Y-bearing spermatozoa was 508:501.     

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.     

A method for cytogenetic analysis of boar spermatozoa using hamster oocytes.

In this paper, the authors detail a method for displaying boar spermatozoa chromosomes using heterospecific zona-free hamster oocyte penetration technique. Semen samples from two Large-White boars having a normal spermogram were studied. The first one had a normal karyotype (38,XY), the second carried a reciprocal translocation rcp(3;7)(p1,3;q2,1). After in vitro fertilization by capacitated sperm, culture and cytogenetic analysis of hamster eggs we obtained metaphase spreads of spermatozoa chromosomes. The ratio of X- and Y-bearing spermatozoa was 49.2% and 50.8%, respectively.     

Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?

It has been reported that the mammalian female could have a preconceptual influence on the sex of her offspring, and it has been hypothesized that this influence could go some way toward accounting for the reported lower fertility following insemination with sex-sorted sperm. To test whether in vitro matured oocytes are able to select X- or Y-bearing spermatozoa following in vitro fertilization (IVF), we fertilized in vitro 1788 oocytes with X-sorted semen, Y-sorted semen, a mix of X- and Y-sorted semen, and unsorted semen from the same bull, and cultured until Day 9. Fertility was assessed by recording cleavage rate at 48 h postinsemination (hpi) and blastocyst development until Day 9. Embryos were sexed at the two- to four-cell stage and the blastocyst stage. The proportion of zygotes cleaving at 48 hpi was not different between X- and Y-sorted groups and the mix of X- and Y-sorted semen group; however, all were significantly lower than the unsorted group (P < 0.001). Blastocyst yield on Day 6 was significantly higher (P < or = 0.01) in the control group compared with the rest of the groups. Cumulative blastocyst yields on Days 7, 8, and 9 were also significantly higher (P < or = 0.01) in the unsorted group compared with the sorted groups. The proportion of female and male two- to four-cell embryos obtained following IVF with X- and Y-sorted sperm was 88% and 89%, respectively and the sex ratio at the two- to four-cell stage was not different following IVF with unsorted or sorted/recombined sperm (56.9% males vs. 57% males, respectively). At the blastocyst stage, similar percentages were obtained. In conclusion, the differences in cleavage and blastocyst development using sorted versus unsorted sperm are not due to the oocyte preferentially selecting sperm of one sex over another, but are more likely due to spermatic damage caused by the sorting procedure.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.     

Selection and separation of X- and Y- chromosome-bearing mammalian sperm

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X- and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X- and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating X-and Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X- and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X- and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X- and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications. Generally accepted experimental laboratory procedures for verification of proportions of X- and Y-bearing sperm are lacking. Staining of sperm with the fluorochrome dye quinacrine will identify a structure known as the “F-body” in human sperm and sperm from a few primates. The dye does not work other mammalian sperm. Its validity as a measure of sperm genotype is the topic of debate. We have used two methods to verify claims of separation of sperm. flow cytometry, and in vitro fusion. One can use flow cytometry to test the efficiency of separation of sperm samples. We tested seven commercial methods for the separation of bovine sper, and none were found of result in enrichment. We also used in vitro fusion of human sperm to denuded hamster ova to test enrichment of Y-bearing sperm from the albumin separation process. out results demonstrated no Y-bearing-sperm enrichment from this process. Scientific problems impeding the success of separation seem to be under investigation with an ever-increasing rate. Hybridization probes for DNA sequences specific to the X or Y chromosome may be the next appropriate technology to test of the selection and separation of X- and Y-chromosome-bearing mammalian sperm.     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Analysis of sperm chromosome complements from a man heterozygous for a pericentric inversion of chromosome 1

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome (1)(p31q12). Q-banded pronuclear chromosomes were analyzed after in vitro penetration of golden hamster oocytes. A total of 159 sperm were examined: 54% bearing the inverted chromosome 1 and 46% the normal chromosome 1. These frequencies are not significantly different from the theoretical 11 ratio. There were no recombinant sperm with duplications or deficiencies, suggesting that a pairing loop failed to form or that crossing-over was suppressed. The frequency of abnormalities unrelated to the inversion was 5% for numerical, 8.8% for structural, 2.5% for numerical and structural, values not significantly different from control donors studied in our lab. The frequencies of X- and Y-bearing sperm were 46% and 54%, respectively, not significantly different from the expected value of 50%. This is the fifth pericentric inversion studied by human sperm chromosome analysis; recombinant chromosomes have been observed in two of the five cases. Some of the factors associated with an increased risk of recombinant sperm appear to be inversion size greater than 30% of the chromosome and chromosome breakpoints in G-light bands.     

Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

Increased frequency of X-bearing sperm in males from an infertility clinic: analysis by two-color fluorescence in situ hybridization

Semen samples from 34 men visiting the Lübeck infertility clinic were investigated using a two-color FISH method to determine the ratio of X- and Y-bearing sperm. The overall ratio was significantly shifted to a preponderance of X-containing sperm. A statistical comparison with seven reports from the literature which included 53 normal probands demonstrated in our patients a significant tendency of a preponderance of X-bearing sperm and significantly less Y-bearing sperm. Furthermore, the Lübeck sperm samples are remarkably more heterogeneous in respect to their variability of X- and Y-bearing spermatozoa than in the other mentioned studies with normal probands. These phenomena have to be evaluated in further studies on groups of infertile males showing similar infertility histories.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Separation of X- and Y-chromosome-bearing murine sperm by free-flow electrophoresis: evaluation of separation using PCR.

The effectiveness of separation of murine X- and Y-bearing sperm by free-flow electrophoresis was evaluated by the polymerase chain reaction (PCR). The ratio of X- and Y-bearing sperm from cauda epididymis was analyzed before and after free-flow electrophoresis. A Y-chromosome-specific sequence (pY353/B) and an autosomal sequence (myogenin) were used to estimate the ratio between X- and Y-sperm in the separated fractions. Cauda epididymal mice sperm were separated into two peak fractions under the electric field. Each peak fraction contained sperm of normal shape, however, the motility of the sperm was extremely diminished after separation by electrophoresis. DNA was extracted from 10(4) sperm from each fraction and from the unseparated sperm, and Y-chromosome specific PCR was performed. The PCR experiment revealed that fraction No. 16 (the peak near the cathode) was a Y-sperm rich fraction, whereas fraction No. 22 (the peak near the anode) was a Y-sperm poor one. These results suggested that murine X- and Y-sperm could be successfully separated by free-flow electrophoresis. Analysis of the chromosome-specific sequence by PCR was demonstrated to be a direct and adequate method to evaluate the separation of X- and Y-sperm.     

体外受精-胚胎移植中完全受精失败的原因分析

受精是生命起源至关重要的一个步骤。在辅助生殖的过程中,完全受精失败发生具有其复杂性和不可预见性。受精失败常伴随着一些胞间调控机制异常,其中,可能阻滞在与精子穿越卵冠丘复合体、精子-透明带结合/穿透、精子-卵膜结合、卵子激活、精子去浓缩或原核形成等任一阶段。通过卵胞浆内单精子注射可以避免大部分受精失败现象,但某些患者仍无法成功受精,即使采用辅助人工激活也无法完全避免其发生。对于在辅助生殖过程中完全受精失败患者,结合其卵子成熟情况、精子质量及相关检测结果,在后续周期调整临床方案可有效避免受精失败的再次发生。     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Gamete rescue in the African black rhinoceros (Diceros bicornis)

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro.