Differential cleavage and developmental rates and their correlation with cell numbers and sex ratios in buffalo embryos generated in vitro

In vitro matured and fertilized buffalo oocytes were co-cultured with buffalo oviductal epithelial cells (BOEC) in CRlaa medium. Cleaved embryos were separated according to the time of completion of first cleavage (i.e., before 30 h and after 30 h post insemination) and cultured for 5 to 10 d and allowed to develop to the blastocyst stage. Zygotes cleaving before 30 h were termed fast-cleaving while those cleaving after 30 h were termed slow-cleaving. The results indicated that fast-cleaving embryos are more likely to develop into blastocysts (25%) than slow-cleaving embryos (7.8%). The quality and viability of fast-cleaving and fast-developing blastocysts was found to be better than that of slow-cleaving, slow-developing blastocysts as judged by cell numbers (67.7 +/- 3.7 vs 35.2 +/- 2.1). However, the mitotic index was not different between the 2 groups. The sex of fast-developing and slow-developing blastocysts was determined via the polymerase chain reaction (PCR) to correlate the rate of embryonic development with the sex ratio of the embryos. Embryos produced by Bull 293 and Bull M-82, irrespective of their being fast or slow-developing, gave rise to more females and males, respectively. From these results, we suggest that there may be a sire effect on sex ratio of in vitro produced buffalo embryos.     

Altered sex ratio of live young after transfer of fast- and slow-developing mouse embryos

We cultured eight-cell mouse embryos to blastocyst stage, divided them into three groups according to the time of blastocoel formation, and transferred them separately into recipients. The proportion of live young from the fast-developing embryos was slightly high (49%) but not significantly different from those of other embryos (38% and 39%). However, the sex ratio of live young from the fast- and slow-developing embryos was significantly shifted toward the male (71%) and to the female (80%), respectively.     

Sex and development in bovine in-vitro fertilized embryos

In vitro fertilized bovine embryos were karyotyped at Day 7 and Day 8 post insemination. The results showed that the percentage of males (sex ratio) was dependent on the developmental stage. Among expanded blastocysts (the most advanced embryos), the sex ratio was 100%, followed by 89% for expanding blastocysts, 75% for blastocysts, and 40% for young blastocysts, all analyzed at Day 7. For embryos karyotyped at Day 8, the sex ratio was 20%. The average mitotic index for all in vitro fertilized embryos was 3.5%. These results suggest that the apparent relationship between sex and developmental rate could be used as a method for noninvasive sexing of in vitro fertilized embryos.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P<0.05), and the highest rate of normal fertilization was observed for 5h culture (73.4%; P<0.05). The sex ratio was significantly (P<0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P<0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%; P<0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1h (treatment 5) or 5h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P<0.05), and the 1h co-incubation significantly skewed (P<0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P<0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly (P<0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.     

Relationship between stage of development and sex of bovine IVM-IVF embryos cultured in vitro versus in the sheep oviduct

We have confirmed more rapid development of male compared with female in vitro-cultured bovine embryos during the first 7 d after in vitro fertilization. The male-to-female sex ratio of expanded blastocysts after 10 d of in vitro culture was 1.37:1.00, which was significantly different from the expected 1:1 ratio, but no deviation from a 1:1 ratio was observed for male and female expanded blastocysts derived from culture of bovine embryos in the sheep oviduct (1.11:1.00). When embryos that developed only to the morula stage were analyzed for sex, a greater number of female than male bovine embryos was observed from in vitro culture but not after culture in the sheep oviduct. Possible causes of these sex-related differences in development of cultured bovine embryos are discussed.     

Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system

In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization.     

Sex of bovine embryos may be related to mothers' preovulatory follicular testosterone

Although the sex of the offspring in mammals is commonly viewed as a matter of chance (depending on whether an X or a Y chromosome-bearing spermatozoon reaches the ovum first), evolutionary biologists have shown that offspring sex ratios are often significantly related to maternal dominance, a characteristic that has been shown to be linked to testosterone in female mammals, including humans. Hence, we hypothesized that variations in female testosterone might be related to reproductive mechanisms associated with sex determination, with higher levels of follicular testosterone being associated with a greater likelihood of conceiving a male. To investigate this hypothesis we collected follicular fluid and cumulus-oocyte complexes from bovine antral follicles. Individual matched samples of follicular fluid were assayed for testosterone, whereas the oocytes were matured, fertilized, and cultured in vitro. The resultant embryos were sexed by PCR. The level of testosterone in the follicular fluid was then compared with sex of the embryo (n = 171). Results showed that follicular testosterone levels were significantly higher for subsequently male embryos (Mann-Whitney U = 2823; P [one-tailed] = 0.016). When we excluded embryos from follicles in which the estradiol-to-testosterone ratio was more than 1 (leaving a sample size of 135), the same result held (Mann-Whitney U = 1667; P [one-tailed] = 0.009). Thus, bovine ova that developed in follicular fluid with high concentrations of testosterone in vivo were significantly more likely to be fertilized by Y chromosome-bearing spermatozoa.     

The sex ratio of bovine embryos produced in vitro in serum-free oviduct cell-conditioned medium is not altered

The sex ratio of bovine blastocysts produced in vitro in serum-free oviduct cell-conditioned medium was investigated. Bovine embryos reaching the blastocyst stage were removed from culture medium on Days 6, 7, 8 and 9 and were identified as small, mid-sized or expanded blastocysts. One third (29/91) of the blastocysts appeared on Day 6. Twelve from them were small blastocysts (5 males), 7 were mid-sized blastocysts (4 males) and 10 were expanded blastocysts (5 males). On Day 7, 33 blastocysts were obtained: 8 small (5 males), 9 mid-sized (3 males) and 16 expanded (13 males) blastocysts. Finally, on Days 8 and 9, 29 blastocysts were obtained: 12 small (9 males), 9 mid-sized (6 males) and 8 (3 males) expanded blastocysts. Sexing of the 91 blastocysts was performed by using an original polymerase chain reaction (PCR) protocol generating discreet internal control signals from both female and male samples and Y-specific smears from the male samples. Proportions of male embryos on Days 6, 7 and on Days 8+9 were 48, 64 and 62%, respectively. These values did not differ significantly among days and did not differ from 50%. Fifty-nine percent of small blastocysts, 52% of mid-sized blastocyst and 62% of expanded blastocysts were male. No difference between these values or with respect to 50% could be observed. These results show that bovine blastocysts produced in serum-free oviduct cell-conditioned medium do not have an altered sex ratio.     

Ped gene expression by embryos cultured in vitro

The rate of cleavage division of preimplantation mouse embryos has been shown to be influenced by the Ped gene, a gene linked to the H-2 complex, the major histocompatibility complex of the mouse. There are two functional alleles of the Ped gene, slow and fast. To examine Ped gene expression outside of the maternal uterine environment, embryos from inbred and congenic mouse strains were cultured in vitro, in chemically defined medium, for various lengths of time. The results of these studies show that the difference in the rate of cleavage division between slow-developing strains (Ped slow) and fast-developing strains (Ped fast) is maintained in vitro. Thus, the Ped gene phenotype of developing embryos is an intrinsic property of the embryos themselves.     

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

Effects of exogenous putrescine on murine preimplantation development in vitro.

After first demonstrating that murine embryos take up putrescine from the medium in which they are cultured in vitro, fertilized eggs were placed in culture and maintained for 4 days in medium supplemented with varying amounts of putrescine. Their development was monitored each day. While embryos that were cultured in putrescine-supplemented medium developed at the same rate as control embryos, a significantly higher percentage of the putrescine-treated embryos attained the blastocyst stage as compared to the control group.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum

The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.     

Influence of the duration of gamete interaction on cleavage,growth rate and sex distribution of in vitro produced bovine embryos

Various factors including the length of gamete interaction and embryo culture conditions are known to influence the rate of development and sex ratio of mammalian embryos produced in vitro. While the duration of gamete interaction deemed optimum would vary depending upon the species involved and the preferred sex in the outcome of in vitro procedures, the mechanisms favoring the selection of embryos of one sex over the other, or the exact time of post-fertilization stage at which a sex-related difference in growth rate is manifested, are not fully understood. In order to determine the optimum length of gamete co-incubation and the impact of male gamete 'aging' on the growth rate and sex ratio of bovine embryos, a series of experiments was carried out using in vitro matured (IVM) oocytes. In experiment 1, IVM oocytes were co-incubated with sperm from two different bulls for 6, 9, 12 and 18 h and the presumptive zygotes were cultured for approximately 7.5 days (178-180 h post-insemination (hpi)) prior to assessing the cleavage rate, blastocyst yield and the sex ratio of blastocysts in each co-incubation group. In experiment 2, the blastocysts obtained from different co-incubation groups were subjected to differential staining to determine the total cell number (TCN) and the proportion of cells allocated to the inner cell mass (ICM) in male and female embryos to test for sex-related differences in cell proliferation or in differentiation of the two embryonic cell lineages in the blastocysts. In experiment 3, IVM oocytes co-incubated for 6, 9, 12 and 18 h with sperm from a single bull, were cultured for 3 days (72 hpi) and the pre-morulae, categorized according to the specific stage of early development, were sexed to determine if a sex-dependent difference is detectable before the blastocyst stage. In experiment 4, IVM oocytes exposed to prolonged co-incubation (18 and 24 h) were compared with those co-incubated with "aged" (pre-incubated) sperm to determine if "aging sperm" is a factor affecting the growth rate and sex ratio of the out come. Our experiments showed that (1) the shortest period (6 h) allowed the highest proportion of cleaved oocytes to reach the blastocyst stage regardless of the semen donor, (2) males out number females (over 2 to 1) among blastocysts when co-incubation of gametes is reduced to 6 h, (3) the male blastocysts display higher total cell count, and (4) the faster growth rate of the male embryos does not affect the early differentiation and allocation of cells to the ICM. Furthermore, our results indicate that the disruption of the expected 1:1 ratio for male and female embryos in the short term co-incubation group is evident as early as the 4-cell stage and peaks at the 8-cell stage and that prolonged gamete interaction tends to reduce the blastocyst yield to even out the sex ratio. Absence of a significant effect on the yield and sex ratio of blastocysts in the prolonged co-incubation groups irrespective of the type of sperm (aged versus non-aged) used suggest that the preponderance of male embryos in short term gamete interaction group may be dependent upon the in vitro advantage of the Y-chromosome bearing sperm. This advantage, manifested in the precocious development during the pre-morulae stage is confined to a short duration that is neutralized when gamete interaction is allowed to proceed beyond 6h.     

Pluripotency of Homozygous-Diploid Mouse Embryos in Chimeras

Pluripotency of mouse uniparental cells (complete homozygous-diploid gynogenetic) produced by embryo manipulation was examined in aggregation chimeras with normally fertilized embryos. A male pronucleus was removed from fertilized eggs by micromanipulation and eggs were diploidized with cytochalasin B. Uniparental cells that developed to 4-cell or more advanced stages were aggregated with normally fertilized 8-cell embryos and transferred to the pseudopregnant female uteri to develop to term. Among the pups, 1 female and 3 males were identified as overt chimeras by their coat color and pigmentation of the retina. Using electophoretic analysis of the isozymes, the contribution of uniparental cells in these chimeras was confirmed by findings in the major organs such as liver, brain, small intestine, kidney, spleen, heart and testis. The female chimera produced offspring derived from oocytes of uniparental origin. Our experiments verified the pluripotency of microsurgically produced mouse uniparental cells.