The Effects of Blastomere Biopsy and Oxygen Tension on Bovine Embryo Development, Rate of Apoptosis and Interferon-τ Secretion

A series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% of embryos proceeding to the blastocyst stage, which was lower than when biopsies were performed at 72 h (37.5%, p < 0.05). In the second experiment, embryos were cultured either under atmospheric or 5% O(2) following blastomere removal. Biopsies had no effect on rate of blastocyst formation with 36% of controls and 33.7% of biopsied embryos proceeding to that stage. However, culture under 5% O(2) significantly increased the number of blastocysts from 29.9% to 40.3% (p < 0.05). This effect was significant in both biopsied and control embryos. In the final experiment, biopsied embryos were again cultured under different oxygen tension. Blastocysts were collected and cultured individually for 48 h in medium droplets in their respective O(2) concentration after which time the medium was assayed for concentration of interferon-tau (IFN-tau). Reduced O(2) concentration again significantly increased blastocyst formation from 24.9% to 41.9% (p < 0.05). IFN-tau secretion was not affected by biopsies, but culture under atmospheric O(2) resulted in significantly increased IFN-tau concentration in medium droplets (12274.0 +/- 2825.9 pM vs 5046.5 +/- 2562.2 pM; p < 0.05).     

Follicle Ablation Improves the Ovarian Response and the Number of Collected Embryos in Superovulated Cows During the Early Stages of Lactation

A field study was designed to compare ovarian response and embryo yield in cows during early lactation when gonadotropin administration followed one of four treatments. In group 1A (n = 19) and 1B (n = 9), the estrouses were synchronized by two prostaglandin F2alpha (PG) injections given 11 days apart, and starting from day 9 of the synchronized cycle superovulation was conducted with eight decreasing dose of FSH. In group 1B, ablation of all follicles >3 mm was carried out on day 8. In group 2A and 2B (each n = 9), a progesterone plus oestradiol intravaginal device (PRID) was inserted for 11 days and gonadotropin administration started on day 9, while cows from group 2B had a follicle ablation on day 8. In all groups, two PG injections were given along with the sixth and the seventh dose of FSH, and the cows were twice inseminated 12 and 24 h after estrus detection. Embryos were collected on day 7. In cumulative results from aspirated and non-aspirated cows, follicular ablation significantly improved: the ovarian response (10 +/- 1.23 vs 6.69 +/- 0.60 corpora lutea per donor), the mean collected embryos (6.57 +/- 0.94 vs 2.46 +/- 0.53) and the mean transferable embryos (4.43 +/- 0.89 vs 2.18 +/- 0.47). Group 1B and 2B cows had better ovarian response than 1A (6.44 +/- 0.81, 12.25 +/- 4.11 and 9.44 +/- 0.93, for groups 1A, 1B and 2B, respectively, p < 0.05). Similarly, from groups 1B and 2B more (p < 0.05) embryos were collected in comparison with their respective group, while the mean transferable embryos from group 2B (5.22 +/- 1.13) was greater (p < 0.05) than that of group 1A (1.67 +/- 0.35), and tented to be greater than those of groups 2A (3.44 +/- 1.19, p = 0.062) and 1B (3.00 +/- 1.78, p = 0.066). The highest (p < 0.05) transferable embryo collection rate was recorded in group 2B (55.29%), followed by that of group 1B (41.33%). In summary, early in lactation, an acceptable number of transferable embryos can be collected from high producing dairy cows, when follicle ablation prior to superovulation is combined with progesterone and oestradiol administration.     

Addition of alpha-tocopherol to culture medium improves the quality and cryosurvival of nuclear-transferred ovine embryos

The aim of this study was to reconstruct and cryopreserve somatic cell nuclear transferred (SCNT) ovine embryos and evaluate the effect of alpha-tocopherol on blastocyst development and subsequent cryosurvival of the SCNT embryos. The alpha-tocopherol (100 microg/ml) was added into culture medium for the SCNT embryos, the yield and total cell numbers of blastocysts were determined and the apoptosis incidences of the blastocysts were evaluated using the TUNEL assay. The blastocysts from the alpha-tocopherol and untreated groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. The results showed that there were no significant differences in blastocyst yield (26.3 vs. 22.3%) and total cell number (68.2 vs. 64.3) between the alpha-tocopherol and untreated groups. However, addition of alpha-tocopherol to the culture medium significantly decreased the apoptotic cell number (3.4 vs. 5.5) and significantly increased the cryosurvival of SCNT blastocysts (66.8 vs. 50.7%). In conclusion, addition of alpha-tocopherol to SCNT embryo culture medium was beneficial for improving embryo quality by decreasing the apoptotic blastocyst cell number and improving the tolerance of the embryos to cryopreservation.     

Relationship between plasminogen activator production and bovine embryo development in vitro

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.     

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.     

小鼠胚胎的玻璃化冷冻研究

用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。     

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.     

Effect of culture medium and prostaglandin I(2) (PGI(2)) analogue on in vitro development of parthenogenetically activated cat oocytes

A method of reliably producing developmentally competent cat embryos in vitro is a prerequisite for study of the physiology of early development and application of assisted reproductive techniques. Oocytes were collected and then cultured in TCM-199 + 10% FBS for 4 h. The matured oocytes were activated with a 20 microsec electric pulse at 1.2 kV/mm. The activated oocytes were incubated in 2 mM of 6-dimethylaminopurine (6-DMAP) for 4 h and were then divided randomly among the treatment groups. In experiment 1, we compared the effects of three culture systems (TCM-199, CR1-aa and Tyrode's) on the in vitro development of parthenogenetically activated cat oocytes. In experiment 2, we investigated the effect of addition of Iloprost (a stable prostaglandin I(2) analogue) to Tyrode's medium on in vitro development of parthenogenetically activated oocytes. As a control, we recovered in vivo produced blastocysts and determined their average cell number. In experiment 1, the cleavage frequency of the oocytes cultured in TCM199, CR1-aa and Tyrode's media were similar (74, 72 and 83%, respectively). However, the incidence of in vitro development to the blastocyst stage was significantly higher in Tyrode's medium (20.4%) than in TCM-199 (2.4%) or CR1-aa (11.1%). Likewise, the average cell number of in vitro activated blastocysts was higher in Tyrode's than in CR1-aa or TCM-199 (106.5 +/- 45.2 vs. 68.3 +/- 25.4 and 35.0 +/- 7.7, respectively; P<0.05). In experiment 2, the percentage of parthenogenetically activated oocytes that underwent in vitro blastocyst development was significantly improved by addition of Iloprost to the culture medium (33.6 vs. 19.1%; P<0.05). The average cell number of in vivo blastocysts (909.0 +/- 226.4) was significantly higher than those of in vitro blastocysts cultured in Tyrode's medium supplemented with or without Iloprost (103.2 +/- 31.3 and 112.2 +/- 39.3, respectively; P<0.05). This result indicated that the current culture method for cat pathogenetically activated oocytes requires further improvement.     

Offspring from non-stimulated calves at an age younger than two months: a preliminary report

This study investigated the viability of embryos from non-stimulated 2-3-month-old calves generated in vitro using oocytes from follicles of defined size in terms of their ability to produce full-term pregnancies. Ablation of follicles>or=4 mm was used to induce the emergence of a new follicular wave, and calves (n=3) were laparoscopically punctured three times at 7-day-intervals to recover cumulus-oocyte-complexes (COCs) from follicles>8 (group A) and between 4-8 mm (group B). Calves were aged 49, 56, and 80 days, respectively, at first recovery. Morphologically intact COCs were subjected to in vitro maturation, fertilization, and embryo culture, and compact morulae/blastocysts were transferred on day 7 post-insemination to synchronized virgin heifers. Blood typing was used for maternity analysis. A total of 29 COCs were recovered, 21 cultured, yielding 11 cleaved embryos (52.4%) and 6 compact morulae/blastocysts (28.6%). No differences were observed between groups. Transfer of the 6 embryos to 5 recipients resulted in three pregnancies (one from group A and two from group B). Two normal male offspring (both from group B), with birth weights of 44 and 51 kg, were born, and two donor calves, aged 56 and 59 days, were identified as the dams. In conclusion, the results demonstrate that embryos generated in vitro from oocytes from non-stimulated calves at an age younger than two months are viable in terms of their ability to produce full-term pregnancies, and suggest that the viability of calf embryos is not related to follicle size.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.     

Effect of potassium simplex optimization medium and NCSU23 supplemented with beta-mercaptoethanol and amino acids of in vitro fertilized porcine embryos

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4- cell (80.8 to 85.4% vs. 73.6%) and blastocyst (30.4 to 30.5 vs. 23.5%) stages and the number of TE (51.4 to 53.8 vs. 35.8) and total cells (67.2 to 71.2 to 48.8) over the control group. On the other hand, supplementing KSOM with AA and/or beta-ME produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4-cell (78.8% vs. 67.7%) and morula (57.8% vs. 46.3%) stages and the number of ICM (18.6 to 19.2 vs. 11.6) and total cells (62.8 to 70.6 vs. 42.8) over control group. In conclusion, our study demonstrates that both KSOM and NCSU-23 medium supplemented with AA and beta-ME and/or only beta-ME alone are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.     

The Effect of Long‐term In Vivo Culture in Bovine Oviduct and Uterus on the Development and Cryo‐tolerance of In Vitro Produced Bovine Embryos

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.     

Susceptibility of bovine naked 2- and 4-cell embryos and hatched blastocysts produced in vitro to infection with noncytopathogenic bovine viral diarrhea virus.

To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.     

Quality and Developmental Ability of Dromedary (Camelus dromedarius) Embryos Obtained by IVM/IVF, In Vivo Matured/IVF or In Vivo Matured/Fertilized Oocytes

The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro fertilized oocytes whether they were matured in vitro (Group 1; 35% and 32%) or in vivo (Group 2; 32% and 45%). Pregnancy rates were not significantly different amongst all groups for the three first months following embryo transfer. All pregnancies were lost after day 90 follow transfer except for in vivo matured and in vivo matured/fertilized groups. Only in vivo matured/in vitro fertilized and in vivo matured/fertilized produced embryos continued normal development until term and resulted in the birth of normal and healthy live calves. Six claves (29%; 6/21) were born from Group 3 and one (8%; 1/13) calf was born from Group 2. This study shows that the IVC system used is able to support camel embryo development. However, developmental competence and viability of dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.     

The effect of gonadotropin releasing hormone on superovulation in elite swamp buffalo cows (Bubalus bubalis)

The effect of gonadotropin releasing hormone (GnRH) supplement was investigated in twenty eight FSH-treated buffalo cows. Animals were assigned to three groups; Group I: GnRH was given at standing heat (n=9), Group II: GnRH was given 8-12 hr after standing heat (n=8) and Group III: Control group with FSH alone (n=11). The responses (no. of corpora lutea and no. of anovulatory follicles), the number of recovered embryos and transferable embryos among the three groups were compared following slaughter of the animals on days 6 to 7 after first mating. The results indicated that the application of GnRH in FSH-treated animals gave no advantage by increasing in the number of ovulations or recovered embryos in all the treatment groups (P>0.05): 4.33 +/- 3.35 vs 3.88 +/- 4.09 vs 4.5 +/- 2.68 for corpora lutea, and 2.33 +/- 2.24 vs 2.0 +/- 3.20 vs 1.91 +/- 2.74 for recovered embryos respectively. GnRH treatment tended to reduce the number of anovulatory follicles but the finding was not significant; 6.11 +/- 3.3 vs 7.38 +/- 4.84 vs 10.18 +/- 2.74 follilcles (P>0.05). The supplementation of GnRH at 8-12 hr after standing heat seemed to produce more transferable embryos than those of treated at standing heat or the controls 1.63 +/- 2.77 vs 1.25 +/- 1.67 vs 1.36 +/- 1.69 embryos respectively.     

Transfer of porcine embryos after 3 days of in vitro culture

Two experiments were conducted to determine the viability of porcine embryos transferred after long-term in vitro culture. In Exp. 1, four-cell embryos were kept in culture for 120 h. Embryos that were exposed to fresh culture medium every 12 h survived better than embryos kept in the same medium throughout the culture period. In Exp. 2, four- and eight-cell embryos were cultured in vitro for 72 h before transfer to estrus-induced recipient gilts. Each gilt received, on average, 19 embryos. If recipients were synchronous with donors 3/32 (9%) recipients remained pregnant with an average of 4.0 +/- .6 viable young. If the sexual cycle of the recipients was 24 h behind that of the donors the pregnancy rate was 18/34 (53%) with 4.4 +/- .5 viable young. Average embryo survival rate for the two groups was 1.8 and 12.5%, respectively. A 24-hourly medium replacement during the in vitro culture period had no significant effect on transfer results. When transferring freshly collected blastocysts, pregnancy rate, number of viable young and survival rate of embryos were 6/10 (60%), 7.8 +/- 1.4, and 23.9% for synchronous recipients and 7/10 (70%), 9.3 +/- 1.8, and 32.9% for asynchronous recipients, respectively. Recipients with very high plasma progesterone levels or numerous follicular cysts at the time of transfer were less likely to remain pregnant than others.     

Experimental transmission of bovine viral diseases by insemination with contaminated semen or during embryo transfer

Three experimental approaches were used to study transmission of blue tongue (BT), infectious bovine rhinotracheitis (IBR) and bovine virus diarrhoea (BVD) viruses. These were insemination with contaminated semen, experimental infection of embryo donor cows, or transfer of embryos experimentally exposed to virus in vitro to normal recipients. Parameters assessed included number and quality of embryos produced, virus detection (isolation and electron microscopy), serology and histopathology. All superovulated sesceptible cows inseminated with semen containing blue tongue virus (BTV) (n = 2) or infectious bovine rhinotracheitis virus (IBRV) (n = 2) became infected. One cow inseminated with semen containing BTV produced seven virus-free seven-day-old embryos; the second cow failed to produce any embryos. One of two cows inseminated with semen containing IBRV produced two underdeveloped, virus-free embryos while no embryos were produced by the second cow. One of two cows inseminated with semen containing bovine viral diarrhoea virus (BVDV) became infected. Two poorly developed, virus-free seven-day-old embryos were recovered from one of these cows. Superovulated susceptible cows inoculated either intramuscularly with BTV (n = 3) or intranasally with IBR virus (n = 2) became infected. Virus was isolated from some tissues of two BTV-infected cows, neither of which produced embryos. A third BTV-infected cow produced two virus-free embryos collected at necropsy five days after inoculation. One of two cows experimentally infected with IBR virus, produced three embryos but virus was not detected either by electron microscopy (1 embryo) or in cell culture by cytopathic alterations (1 embryo).(ABSTRACT TRUNCATED AT 400 WORDS)