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This protocol summarises zona-free sheep cloning by somatic cell transfer (SCT), adapted from a similar SCT method in cattle (https://doi.org/10.1089/153623003772032763). It describes the workflow that is required for reprogramming somatic cells into cloned blastocysts of high morphological quality, including two optional steps of embryo aggregation and vitrification. The resulting embryos can then be transferred into surrogate ewes for producing live cloned animals. Compared to conventional zona-intact procedures, this zona-free embryo reconstruction system increases throughput of cloned embryo production and ease of operation.

The present study aimed to establish a zona free (handmade cloning) embryo production system for Pashmina goat embryos. Abattoir derived oocytes were matured in vitro; after maturation, oocytes were enucleated and fused with somatic cells derived from an adult Pashmina goat tissue. The reconstructs were activated using a calcium ionophore-DMAP procedure. The embryos were distributed into two experimental groups. In Experiment 1, the embryos were cultured in one of the following four culture media (i) G1.G2 media (ii) Modified synthetic oviduct fluid (mSOF) (iii) Research vitro cleave media (RVCL) and (iv) Embryo development media (EDM), and were cultured for 7 days. The cleavage rates in G1.G2, RVCL, and mSOF were higher than those in EDM (86.8, 82.4, 77.3, and 68.8%, respectively). Blastocyst rates were higher in RVCL than those in mSOF, EDM, and G1.G2 (15.0, 10.5, 4.9, and 2.2%, respectively). In experiment 2, the embryos were cultured in five different culture systems: (i) Flat surface (FS), (ii) Well in drop (WID), (iii) Well of well (WOW), (iv) Micro drop, and (v) Hanging drop, for 7 days. The cleavage rates in FS and WID were higher than those in WOW, Micro drop, and Hanging drop (84.3, 81.2, 73.6, 73.5, and 70.3%, respectively). The blastocyst rates were higher in WID than those in WOW, Micro drop, Hanging drop, and FS systems (21.6, 13.7, 11.5, 10.9, and 3.9%, respectively). The embryos produced in experiment 2 were transferred to synchronized recipients. Of the three pregnancies established on day 40, one resulted in the delivery of a healthy Pashmina kid.

Evaluation of sperm function provides finality to the battery of in vitro assays while discriminating between a good from that of bad quality semen sample. In this important chapter, we have described zona binding assay with four types of variations, namely, hemizona, zona free, heterologous and homologous zona binding assays. Merits and demerits of these assays have been discussed here.

The gradual decline in the genetic diversity of farm animals has threatened their survival and risk of their extinction has increased many fold in the recent past. Endangered species could be rescued using interspecies embryo production. The objective of this study was to investigate the effect of three different culture media on the development of Handmade cloned intraspecies (goat-goat) and interspecies (goat-sheep) embryo reconstructs. Research vitro cleave media (RVCL) yielded higher cleavage and morula-blastocyst development in intraspecies and interspecies nuclear transfer groups compared with G1.G2 and modified synthetic oviductal fluid (mSOFaaci). Cleavage frequency of intraspecies cloned embryos in RVCL, mSOFaaci, and G1.G2 did not differ significantly (87.12%, 82.45%, and 92.52%, respectively). However, the morula/blastocyst frequency in RVCL was greater in mSOFaaci and G1.G2 (51.18% vs. 38.28% vs. 36.50%, respectively). Cleavage and morula/blastocyst frequency in interspecies cloned embryos was greater in RVCL than in mSOFaaci and G1.G2 (76.14% and 42.3% vs. 65.9% and 38.3% vs. 58.56% and 33.1%, respectively). Goat oocytes were parthenogenetically activated and cultured in RVCL, mSOFaaci, and G1.G2 and kept as control. Cleavage and morula/blastocyst frequency in this group was greater in RVCL than in mSOFaaci and G1.G2 (89.66% and 65.26% vs. 85.44% and 48.05% vs. 86.58% and 42.06%, respectively). Conclusively, the results suggest that not only can the interspecies embryos of goat be produced using sheep oocytes as donor cytoplast but also the percentages can be improved by using RVCL media for culturing of the embryos.

Studies on parthenogenetic activation of oocytes are important to improve the efficiency of nuclear transfer as artificial activation of oocytes is an essential component of nuclear transfer protocol. The present study was carried out to investigate the effects of different activation methods, culture systems and culture media on in vitro development of zona-free and with-zona parthenogenetic embryos in goat. In case of zona-free parthenogenesis, there was a significant (p

The human spermatozoa zona-free hamster ova penetration assay (SPA) and seminal fluid analysis (SFA) were performed on 44 infertile and 35 fertile couples. The mean penetration rate for semen samples from fertile males (44% ± 3%) was significantly higher (p 0.05) was observed between the spermatozoal density, total spermatozoal count, percentage of motile spermatozoa, or percentage of normal morphology and the penetration rate. The sensitivity of the SPA was 91% and the specificity was 100% as compared to 73% and 83% respectively for the SFA. These findings indicate that the SPA is useful in the assessment of the fertility potential of human semen samples in the evaluation of male fertility and infertility.

In unselected IVF patients, zona-free and laser zona-assisted hatching produced a comparable effect on blastocyst transfer.

Summary Fumarase activity, sperm count, sperm motility and in vitro fertilizing ability of spermatozoa were determined in semen samples of 69 men attending an infertility clinic. There existed a significant correlation between sperm count and fumarase activity (r = 0.52). No significant relationship was observed between sperm motility or hamster egg fertilizing ability of the spermatozoa and fumarase activity. From these results it is concluded that fumarase activity in human ejaculates can probably not be used to define the fertilizing ability of spermatozoa. Fumarase-Aktivitat im menschlichen Ejakulat Beziehungen zwischen Spermatozoen-Motilitat oder -Penetration in Zona-freie-Hamster-Eier Zusammenfassung An den Spermaproben von 69 Mannern wurde die Fumarase-Aktivitat, die Spermatozoendichte, die Spermatozoenmotilitat und die in-vitro-Fertilisierungs-Fahigkeit von Spermatozoen untersucht. Dabei konnte eine signifikante Beziehung zwischen der Spermatozoendichte und der Fumarase-Aktivitat (r = 0,52) festgestellt werden. Zwischen der Spermatozoenmotilitat oder der Fertilisierungs-Fahigkeit menschlicher Spermatozoen fur Hamster-Eier lies sich dagegen keine Signifikanz zur Fumarase-Aktivitat herstellen. Aus den Ergebnissen wird die Schlusfolgerung gezogen, das die Fumarase-Aktivitat im menschlichen Ejakulat im allgemeinen nicht fur die Beurteilung der Fertilisierungs-Fahigkeit der Spermatozoen herangezogen werden kann.

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Several breakthroughs in nuclear transfer research were first achieved in sheep, although cattle soon became the main livestock species of interest. However, sheep still offer significant advantages both in basic and applied research. With increased interest in cloning of livestock, new approaches have been developed for both sheep and cattle nuclear transfer technology. These include methods for zona-free nuclear transfer that can be performed with or without the use of micromanipulator. Here we describe four different nuclear transfer methods including the traditional micromanipulation-assisted method in sheep, zona-free method in sheep in which the order of enucleation and nucleus delivery have been reversed ("reverse-order" cloning) and zona free manual cloning methods ("hand-made cloning") for embryonic and somatic cloning in cattle. The purpose of this paper is to encourage people to familiarize themselves with these different methods available and to help them choose and test the method most suitable for their particular circumstances.

In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 μM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 μM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 μM ionomycin for 5 min rather than 2.5 μM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.

Frozen-thawed mouse embryos with (+ZP) and without (-ZP) zona pellucida have been studied at the Scanning Electron Microscope (SEM) to determine how the process affects the plasma membrane and the subsequent embryo development. The main difference observed in -ZP embryos immediately after thawing is the abnormal morphology and distribution of microvilli. This could explain the spontaneous separation of blastomeres in -ZP embryos, and the decrease in their survival rate. If thawed -ZP embryos are allowed to recover in culture, their plasma membrane characteristics and survival rate are identical to those of control embryos.

Zona-free uncompacted 8-cell (8C) or partially compacted 8-cell (8-M) stage embryos were cocultured with frozen-thawed TT2 embryonic stem (ES) cells at a density of either 5.5 × 105 or 5.5 × 106 cells/ml. When zona-free embryos at the 8C and 8-M stage were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, 5% and 32% of the embryos developed to the blastocyst stage after overnight culture, respectively. At a density of 5.5 × 106 cells/ml, however, developmental rates to the blastocyst stage of 8C and 8-M were only 12% and 15%, respectively. When the 8C and 8-M embryos were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, there was no difference in the number of ES cells attached to either of them. The 8-M showed no significant difference in development to blastocyst stage in spite of the number of attached ES cells. However, the development rate to blastocyst was significantly reduced when the 8C aggregated with high numbers of ES cells. When the blastocysts were transferred into recipients, the percentage of chimeric mice in newborns ranged between 0-25%. In comparison, the percentage of newborn chimeric mice after transfer of the morula to small blastocyst ranged between 13-60%. One cause of this high frequency of chimerism after transferring morula derived from 8C was delayed preimplantation development of the embryos with high numbers of attached ES cells.

The need of the zona pellucida (ZP) for in vitro development is controversial because it might be an obstacle to hatching of the blastocyst. This study investigated the development and quality of porcine parthenogenetically activated (PA) embryos by observation of the developmental kinetics, the developmental percentages, the frequency of apoptosis, and robustness after removal of the ZP by pronase. Three experiments were made between zona-free PA embryos and zona-intact embryos: (1) determination of the timing of developmental stages using time-lapse observations for 6 days; (2) determination of developmental percentages and occurrence of apoptosis on Day 6 and Day 7 (Time of PA, Day 0); and (3) investigation of the robustness of embryos using vitrification on Day 4. The developmental kinetics showed that there was a general trend for zona-free PA embryos to develop faster than zona intact PA embryos at all developmental stages, but the difference was only significant at the five-cell stage. When compared with development of zona-intact embryos, ZP removal decreased the overall blastocyst percentage (83.9 ± 2.0 vs. 72.5 ± 2.9, respectively) and especially the percentage of good morphology (grades 1 and 2 combined) blastocysts (69.5 ± 2.0 vs. 55.7 ± 2.4, respectively). However, the process showed a significant decrease in apoptosis indicating an increased embryonic quality. Still, the survival percentage of porcine PA embryos after vitrification was dramatically decreased after ZP removal at all observation times (P < 0.05). In conclusion, removal of the zona pellucida might improve the embryonic quality by accelerating the speed of embryonic development and decreasing the number of apoptotic cells in blastocysts even though developmental percentages and embryonic robustness might be decreased.

The objectives of the present study are to characterize the motility and penetrability into zona-free hamster eggs of boar spermatozoa from 7 regions of the epididymis. Spermatozoa from the caput epididymidis were immotile or exhibited a flagellant or circular type of movement. However, 80% of the cells from the proximal cauda showed an intensive, forward movement. When the spermatozoa from each region of the epididymis were coincubated with zona-free hamster eggs after preincubation for inducing the acrosome reaction, they exhibited high penetration rates (95-100%) and average number of spermatozoa (3.4-5.8) in penetrated eggs irrespective of their origin. These findings indicate that most of the spermatozoa acquire the ability to exhibit progressive motility during their transit through the corpus, and that at least some of the cells located in the proximal caput have the ability to fuse with egg plasma membrane.

Horse cloning has several interesting applications, but its efficiency remains low. Embryo aggregation has proven to improve cloning efficiency in the mouse (1), bovine (2) and in the equine (3). Aggregation of two and three zona free cloned reconstructed embryos previously demonstrated to result in healthy cloned foals, and the development efficiency of aggregated clones increased proportionally with the number of aggregated structures, resulting aggregation of three embryos better than two, and two better than no aggregation The goal of the present study was to determine the maximum number of embryos that can be aggregated without affecting cloning efficiency through the study of the in vivo embryo development after embryo transfer, the blastocyst size, the in vitro embryo development until day 16, and the apoptosis rate of blastocysts through TUNEL assay.

The correlation between the hypo-osmotic swelling test, the modified Eliasson score for human semen analysis and the hamster egg penetration assay was examined. The results showed a weak but significant correlation between the group of subjects with swelling rates below 50% and the above 80% group. These groups showed hamster egg penetration assay rates of 9 +/- 14% and 39 +/- 29%, respectively (P less than 0.035). A significant correlation was also found between modified Eliasson scores (17 +/- 10 and 5 +/- 7, respectively) and swelling rates below 50% and above 70% (P less than 0.001). These results suggest that spermatozoa showing a low swelling rate have a poor fertilizing capacity. Therefore, the swelling tests and analysis of the patients semen can be used to discriminate sperm quality.

An experiment was conducted to assess the sperm fertility of Sirohi bucks. Ejaculates from three bucks were collected through artificial vagina and evaluated microscopically at 37° C. After final washing with Biggers-Whitten Whittingham (BWW) medium, semen was kept in a CO2 incubator for 20 min (swim up). The hamster oocytes were recovered following superovulation and visualized on the basis of cumulus cell mass. The oocytes were washed in BWW medium and then denuded with 0.1% hyaluronidase, the cumulus-free oocytes were treated with 0.1% protease for 2–3 min for zona lysis. Oocytes and swim up motile spermatozoa were co-incubated at 37° C for 3 h. in already incubated BWW drop (50 μl). At the end of co-incubation, washing of oocyte sperm complexes was done with BWW medium and then kept in hypotonic KCl solution (50 mg/ml), fixed in 2.5% glutaraldehyde for 10 min and stained with 0.1μg/ml DAPI (4,6-di amidino 2-phenyl indole). The number of sperms penetrated into the oocytes was determined. The result of the study indicated that among the three bucks, buck No. 1 had higher penetration rate (100%) as compared to buck No. 2 (93.00%) and buck No 3 (86.66%).

Progesterone plays an important role in sperm capacitation and acrosome reaction. In this experiment we examined the effect of a progesterone antagonist, RU486, on the penetration of zona-free hamster eggs by human sperm. Media containing 10, 15, 20, 25 or 30 μM RU486 were prepared. In the control medium without RU486, the sperm penetration rate was 80.7%. In the medium with 10 μM RU486, the sperm penetration rate was 67.0%. The rate further decreased as the level of RU486 increased. In the medium with 30 μM RU486, the sperm penetration rate was reduced to 4.8%. This study demonstrates that RU486 inhibits penetration of zona-free hamster eggs by human sperm in a dose-dependent manner.

BACKGROUND: The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter

To minimize the risk of identical twinning the zona pellucida (ZP) was completely removed before blastocyst ET in all blastocyst transfers performed between May 1, 1999 and April 30, 2000. Compared to prior, zona intact, blastocyst transfers carried over between September 1, 1998 and April 30, 1999 no significant difference was noted regarding implantation, pregnancy, monozygotic pregnancy, or multiple gestation rates, thereby indicating that monozygosity can arise independently of the ZP.

To investigate the effect of preincubation of human sperm in milk on their ability to penetrate zona-free hamster oocytes, 27 ejaculates were studied. Each ejaculate was divided into two portions and incubated in either milk or TEST yolk (TESTY) for 22-24 hr at 5 degrees C prior to processing for the sperm penetration assay.Spermatozoa preincubated in milk penetrated a significantly (P0.05) higher percentage of oocytes (mean +/- SE; 74.4 +/- 6.3) than spermatozoa preincubated in TESTY (66.8 +/- 6.6). Statistically, no significant differences were noted for penetration index or postincubation sperm motility between the two media.Preincubation of sperm in milk therefore appears to enhance their ability to penetrate zona-free hamster oocytes, compared to TESTY preincubation. Since milk is natural, simple, inexpensive, readily available, and can be easily processed, it should be the medium of choice for sperm preincubation.

Objective To examine the reliability of the zona-free aged (1 day old) human unfertilized oocyte sperm penetration assay for assessing sperm fertilizing ability and to determine the predictive value of this assay for subsequent subzonal insemination (SUZI) outcomes. Design A total of 253 unfertilized oocytes from total fertilization failure patients and from good fertilization rate (>70%) patients in standard IVF were inseminated with donors' spermatozoa, and penetration rates were compared in each group. Two hundred seventy-two unfertilized oocytes from total fertilization failure, poor fertilization ( 30%) were inseminated with husbands' spermatozoa, and penetration rates were compared between the three groups. In 29 patients, the results of the zona-free assay performed in previous IVF were compared with the fertilization rates of subsequent SUZI. Results In the zona-free assay using donors' spermatozoa, there was no difference in penetration rates between the two groups (109/122, 89.3% versus 114/131, 87.0%). Penetration rates using partners' spermatozoa were positively correlated with fertilization rates in standard IVF (total fertilization failure 34/75, 45.3%; poor fertilization 56/77, 72.7%; normal fertilization 108/120, 90.0%). There was a significant difference in fertilization rates after SUZI between the patients with negative and positive penetration in zona-free assay (4/53, 7.5%, versus 54/174, 31.0%). Conclusion The zona-free human oocyte assay may primarily reflect sperm fertilizing ability. This assay also could be a reliable predictor for subsequent SUZI outcome.

A previous study from this laboratory showed that nicotine in vitro has deleterious effects on sperm motion characteristics. This study was conducted to evaluate the effects of nicotine on the ability of human spermatozoa to attach and penetrate zona-free hamster eggs. Spermatozoa from fertile donors, washed free of seminal plasma, were incubated with medium (control) and 0.1, 1, 5, and 10 mM concentrations of nicotine (concentrations estimated to approximate residual concentrations of nicotine in the testes of heavy smokers) for 18 h at 37 degrees C in a humid 5% carbon dioxide incubator. The sperm preparations were then mixed with enzymatically denuded hamster eggs and incubated for 3 h at 37 degrees C. The oocytes were examined by phase-contrast microscopy to enumerate the rates of sperm attachment and penetration. The data were analyzed by a paired t test and repeated measures analysis of variance using the arcsine transformation of the percentages. The percentages of eggs with attached spermatozoa significantly declined in a dose-dependent manner, the highest inhibition being at 10 mM (F = 24). The rate of sperm penetration was even more significantly decreased with the increase in nicotine concentrations in the following order: 10 mM (F = 56)5 mM (F = 30)1 mM (F = 44)0.1 mM (F = 12). Nicotine concentrations of 0.1 mM and above negatively affected sperm penetration of zona-free hamster eggs.

A conditioned medium (CM) was prepared by culturing human sperm at 3.0 x 10(6) sperm/ml for 12 h. The fusion index increased significantly when the sperm penetration assay (SPA) was performed with CM (0.6 without CM to 2.0 with CM). The conditioned medium was also effective on the sperm from 11 patients who showed a low or zero value in SPA and who's in vitro fertilization treatment failed. When CM from the sperm of healthy donors was added to these patients' sperm, the fusing ability to zona free hamster eggs was significantly restored.

Gustafsson H., U. Jaakma and M. Shamsuddin: Viability of fresh and frozen-thawed biopsied bovine embryos. Acta vet. scand. 1994,35,217-222.– Bovine embryos were biopsied using a simplified splitting technique and frozen-thawed according to a standard method with glycerol as cryoprotectant. The viability of fresh and frozen-thawed biopsied and intact embryos were evaluated after in vitro culture, by means of fluorescence test or following transfer to recipients. The survival rates after in vitro culture of fresh intact and biopsied embryos and of frozen-thawed intact and zona free embryos were not significantly different (70%, 60%, 68% and 52%, respectively), but significantly reduced for bipsied frozen-thawed embryos (16%) (p≤0.05). The pregnancy results after transfer of biopsied frozen-thawed embryos were also significantly lower (8%) compared to fresh biopsied embryos (39%) (p≤0.05). Both intact and biopsied embryos fluoresced after incubation with diacetylfluorescin but with higher intensity for the intact embryos. It is suggested that the reduced survivability for the frozen-thawed biopsied embryos might be caused by combined effects of the loss of the zona pellucida and the reduction of cells as a result of the simplified biopsy technique. It is concluded that improved biopsy and/or freezing techniques must be used if biopsied embryos have to be frozen.

The effect of modified incubation systems on the development capacity of the zona-free rabbit embryos was examined. Embryos at 1-, 2- and 4-cell stages were used. The removal of the zona pellucida was accomplished by the enzymic-mechanical technique. Denuded rabbit embryos were cultured using 3 incubation systems. In the first and the second system the embryos were cultured in microdrops. The difference between these first 2 systems concerned the volume of the microdrops and the kind of paraffin oil used. In the first system the embryos were cultured in 5μl microdrops covered with light or heavy paraffin oil; in the second system embryos were cultured in 40-μl microdrops under light paraffin oil. The third traditional system involved the incubation of embryos in glass capillaries into separated columns of medium. The percentage of blastocysts obtained from 1-cell embryos cultured in the first incubation system was 6.1% with heavy paraffin oil as the covering layer and 29.0% with light paraffin oil. In the second and third incubation systems blastocyst yield was 30.8 and 59.6%, respectively. The percentage of blastocysts obtained from 2-cell and 4-cell stage embryos with heavy paraffin oil was 18.7 and 25.0%, respectively; with light paraffin oil these figures were 40.0 and 50.0%, respectively. In the second incubation system these figures were 49.3 and 72.3%; and in the third incubation system they were 72.9 and 78.3%, respectively. The results of the experiment showed that culture into glass capillaries is undoubtedly an effecient method of culturing of the zona-free rabbit embryos.

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.

The method for chromosome preparation from preimplantation mouse embryos devised by Tarkowski (Tarkowski, 1966) is widely used in experimental and clinical cytogenetics. The main advantage of this method is simplicity, but considerable skill is needed to obtain high quality preparation. Moreover, it is difficult to use this method when chromosomal preparations are made from zona free embryos or isolated blastomeres obtained by biopsy of preimplantation embryos. In this paper, the method suitable for the above mentioned materials obtained from different animal species (mice, rats, rabbits, monkeys, etc.) as well as humans is presented. This method was successfully used for karyotyping isolated blastomeres from the four-cell stage mouse embryos (Severova, Dyban, 1984) and the eight-cell stage human embryos (Handyside, 1989).

1. The quality of gamma irradiated spermatozoa from cockerels was investigated by measuring motility, oxygen consumption and ability to penetrate zona‐free hamster ova. 2. Doses of 750 Gy slightly reduced motility and oxygen consumption; however, irradiated spermatozoa penetrated on average 56% (minimum 33%) of hamster ova. 3. The zona‐free hamster ova test may be useful for estimating avian sperm quality.

Endotoxin has been found to be a contaminant in human in vitro fertilization (IVF), leading to low pregnancy rates, mostly through embryo fragmentation. We wished to determine if standard quality control assays (one-cell and two-cell mouse embryo culture) were able to detect the presence of endotoxin and how they compared with the zona-free mouse embryo assays. Endotoxin was detected by the zona-free mouse embryo assays but not by the one- or two-cell mouse embryo assays. When excessive embryo fragmentation is noticed in the human IVF system, the use of zona-free mouse embryo assays is recommended.

The possible effects of N2,2'-O-dibutyryl 3'-5'-cyclic guanosine monophosphate (dbcGMP), imidazole (IM) or their combination on human spermatozoal motility and penetration of zona-free hamster oocytes were investigated. Motility of human spermatozoa was assessed at 0, 0.5, 2, 3, 5 and 8 h over an 8-h incubation period with various concentrations of dbcGMP, IM or dbcGMP plus IM. Both percentage motile spermatozoa and progression of human spermatozoa decreased with increasing concentrations of dbcGMP (12-96 mM) and IM (10-80 mM). This inhibitory effect on motility was more pronounced when dbcGMP was added with IM. For assessment of human spermatozoal penetration into zona-free hamster oocytes, spermatozoa were treated for 30 min with either dbcGMP, IM or both after 5 h of capacitation pre-incubation at 37 degrees C. Alternatively, the compounds were only added during the capacitation preincubation period. At concentrations of dbcGMP less than or equal to 24 mM, neither of the treatments had a significant effect on the penetration rate of zona-free hamster oocytes in comparison with the controls. However, a higher concentration of dbcGMP (48 mM) inhibited the penetration rate. A similar trend was observed when dbcGMP plus IM were added. IM alone had no significant effect on the penetration rate at the concentrations tested (10-40 mM). Our data indicated an inhibitory effect of dbcGMP on both human spermatozoal motility and penetration into zona-free hamster oocytes.

Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.