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.     

Taurine and human spermatozoal capacitation

The effect of taurine at various concentrations (0.01, 0.1 and 1 mM) on the in vitro motility and fertilizing capacity of human spermatozoa was studied. Spermatozoa collected from 10 normal men were washed in BWW medium and incubated with taurine for 5 hours, the period required for spermatozoal capacitation. The percent motilities were recorded at 0 and 5 hours during capacitation preincubation with taurine. After incubation, the spermatozoa were washed with BWW medium to remove taurine before insemination of the zona-free hamster ova for an assessment of the fertilizing capacity. Taurine caused a significant dose-dependent increase in the penetration of the zona-free hamster ova in comparison to the control (p less than 0.05). Taurine did not have any significant effects on the spermatozoal motility during capacitation preincubation. The results suggest that there may be a physiological role for this beta-amino acid in human spermatozoal capacitation in vivo.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

Evaluation of relative fertility of cryopreserved goat sperm

This study was designed to compare differences in the in vivo fertility of cryopreserved goat semen assessed by heterospermic insemination with differences in in vitro analyses. Five groups of does were inseminated with mixed frozen-thawed semen from different pairs of bucks. The percentage of offspring sired by each buck in the pair was compared with the relative ability of spermatozoa from that frozen-thawed ejaculate to penetrate zona-free hamster ova, relative post-thaw acrosomal integrity, ability to undergo an acrosome reaction during in vitro capacitation, and assessments of sperm motility. In 4 of the 5 different insemination pairs, the ratio of offspring born was other than 1:1. Acrosomal integrity, ability of spermatozoa to undergo an acrosome reaction, and parameters of sperm motility were not correlated with differences in relative fertility in this experiment using ejaculates from fertile bucks. The ability of spermatozoa to fuse with the oocyte plasma membrane was highly correlated with relative in vivo fertility (R(2) = 0.78, P = 0.04). This suggests that fusion with the oocyte plasma membrane is an event in the fertilization process in which significant variation exists among fertile bucks. Assessment of ability of spermatozoa to fuse with zona-free hamster ova may contribute to analysis of post-thaw fertility of frozen-thawed buck semen.     

An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova

We have developed an improved method for analyzing human sperm chromosome, using zona-free hamster ova. Our main improvements of methodology are as follows: (1) Fertilization rate of hamster oocytes by human spermatozoa was markedly raised by successive treatments of the spermatozoa with 5-15 microM ionophore A23187 solutions and a capacitation medium (BWW medium) containing 3.5% HSA. The HSA most effective in inducing capacitation was selected from several kinds of HSA products commercially available. (2) Monospermic fertilization was ensured by inseminating oocytes with highly capacitated spermatozoa at a low concentration for a short time. (3) TC medium 199 was used for postinsemination culture of the eggs. (4) A medium containing podophyllotoxin and vinblastine (0.04 micrograms/ml each) was used to block karyogamy and first-cleavage spindle formation. (5) Chromosome slides were prepared with our gradual fixation-air-dry method instead of Tarkowski's method. Ninety-two to 177 spermatozoa corresponding in number to 43%-79% (mean: 62%) of the inseminated oocytes were successfully karyotyped in each experiment. In spite of above-mentioned quantitative improvements, quality of Q-banding was not necessarily satisfactory in our slides. Improvement of banding technique is an important problem to be solved in our method. Spontaneous incidence of chromosome aberrations was studied in a total of 1,091 spermatozoa obtained from nine semen samples from four donors. Incidences of aneuploidy and structural anomaly were 0.9% (hyperhaploidy, 0.45%; hypohaploidy, 0.45%) and 13.0%, respectively. Structural aberrations included breaks (45.1%), fragments (32.4%), exchanges (21.8%), and deletions (0.7%). Ratio of X-sperm to Y-sperm was 53% to 47%. These results were discussed in comparison with those reported previously.     

Use of zona-free hamster ova to assess sperm fertilizing ability of bull and stallion

Zona-free hamster ova interacted with bull and stallion spermatozoa after treatment of ejaculated semen to capacitate the sperm cells. Sperm conditioning by prolonged incubation in BWW medium (18–26 hr) prior to insemination was effective for capacitation of bull and stallion sperm. Preincubation of bull sperm for 70 or 105 min in defined medium (DM) with NaCl content elevated to result in 350 m0sM/kg also led to penetration of hamster vitelli. More rapid sperm conditioning was possible and higher proportions of interacting vitelli followed insemination with bull or stallion sperm exposed to high ionic strength DM (380–390 m0sM/kg) for 10 min before incubation in isotonic DM prior to insemination, the treatment adopted in subsequent work. Initial efforts to assess relative fertilizing ability of freshly ejaculated semen from two fertile bulls (A and B) in A1 usage resulted in uniformly high ( > 90%) levels of sperm-vitelli interaction (for both) when the hamster ova employed resulted from superovulation with PMSG and HCG. Following use of ova from untreated hamsters sperm samples of bull A and bull B interacted with 53.8% and 84.9% (P < 0.05) of zona-free hamster ova, respectively. Conception data (60–90 day nonreturn rates) resulting from A1 with semen collected during the same interval but processed and stored in liquid nitrogen prior to use revealed an inverse relationship to proportions of vitelli interacting with fresh sperm; nonreturn rates were 69.3% and 66.3% for bull A and bull B, respectively. A similar treatment effected capacitation of frozen-stored bull semen to enable sperm-vitelli interaction. These findings encourage additional efforts to correlate testing of processed semen with fertility.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

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.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.     

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg-sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37 degrees C results in significantly higher penetration rates than does a 22 degrees C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen-thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze-thaw procedure results in a shortening of the required capacitation time.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.     

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.     

Ethanol inhibits human and hamster sperm penetration of eggs

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.     

Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova

The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0–24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

Microisolation and microcloning of bovine X-chromosomes for identification of sorted buffalo (Bubalus bubalis) spermatozoa

Flow-cytometry sorting technology has been successfully used to separate the X- and Y-chromosome bearing spermatozoa for production of sex-preselected buffalo. However, an independent technique should be employed to validate the sorting accuracy. In the present study, X-chromosomes of bovine were micro-dissected from the metaphase spreads by using glass needles. Then X-chromosomes were then amplified by PCR and labelled with Cy3-dUTP for use as a probe in hybridization of the unsorted and sorted buffalo spermatozoa -chromosome. The results revealed that 47.7% (594/1246) of the unsorted buffalo spermatozoa were positive for X- chromosome probe, which was conformed to the sex ratio in buffalo (X:Y spermatozoa=1:1); 9.6% (275/2869) of the Y-sorted buffalo spermatozoa and 86.1% (1529/1776) of the X-sorted buffalo spermatozoa showed strong X-chromosome FISH signals. Flow cytometer re-analysis revealed that the proportions of X- and Y-bearing spermatozoa in the sorted X and Y semen was 89.6% and 86.7%, respectively. There were no significant differences between results assayed by flow-cytometry re-analysis and by FISH in this study. In conclusion, FISH probe derived from bovine X- chromosomes could be used to verify the purity of X and Y sorted spermatozoa in buffalo.     

Failure of human spermatozoa to penetrate zona free mouse and rat ova in vitro

When incubated for 8 to 26 hours with zona-free mouse or rat ova, human spermatozoa failed to attach to or penetrate any of the ova. The ova were capable of being fertilized since both intra- and inter-species penetration of spermatozoa and formation of pronuclei occurred between rat and mouse gametes. When mouse spermatozoa were incubated for three to eight hours with rat ova, a high proportion of the ova were penetrated, formation of pronuclei occurred and in 9 out of 36 ova incubated for 40 hours after insemination, regular cleavage and formation of morphologically normal 2-cell embryos occurred. Human spermatozoa retained their morphological integrity and motility only when the culture medium contained purified bovine serum albumin (3 mg/ml) or human serum (5% v/v) and not when unpurified BSA from several different commercial sources was used as a protein source. In this latter medium, the ova of both rats and mice degenerated after 8-hour incubation in the presence of human spermatozoa but not when human spermatozoa were absent or in the presence of either rat or mouse spermatozoa. Electron microscopy indicated that the human spermatozoa incubated for eight hours in medium containing purified BSA had undergone an acrosome reaction. These spermatozoa also attached to and penetrated human oocytes which had been matured in vitro.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Human sperm-cervical mucus interaction and the ability of spermatozoa to fuse with zona-free hamster oocytes

Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4 degrees C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P less than 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.