Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen

During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39 degrees C under humidified 5% CO(2), 5% O(2), 90% N(2) and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.     

In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus

Bovine viral diarrhea virus infection was induced in 16 heifers by inoculation of a noncytopathic strain of bovine viral diarrhea virus (BVDV). Six BVDV-free heifers served as controls. On Day 8 after inoculation, cumulus—oocyte complexes were collected from ovaries of animals at the second peak of fever preceded by leukopenia. The oocytes were then matured and fertilized in vitro. There was no significant difference (48% vs. 54% P>0.05) in the percentage of cleaved oocytes between infected and non-infected animals. However, the proportion of embryos that developed to the blastocyst stage was significantly higher for the control group than for BVDV group (29% vs. 14%) (P<0.01). All follicular fluids and cumulus—oocyte complexes collected from infected animals tested positive for the presence of the virus, but embryos produced by in vitro fertilization 7 days after in vitro co-culture tested negative.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post co?tum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.     

In vitro production of bovine embryos using sex-sorted sperm

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes (N=3526) were aspirated from these ovaries, fertilized 22+/-0.2h later, and cultured to the morula or blastocyst stage. The fluorescence-activated cell sorting ("Beltsville") approach was used to produce (primarily) X-bearing sperm from the ejaculates of three young Holstein sires, and 365 transferable embryos were produced. On average, 3.6+/-0.3 (means+/-S.E.M.) transferable embryos were produced per donor, including 1.4+/-0.2 (Grade 1), 1.5+/-0.2 (Grade 2), and 0.7+/-0.1 (Grade 3) embryos. Number of usable oocytes per donor (33.9+/-3.3) and percent cleavage (51.1+/-1.9) were significant predictors of the number of blastocysts that developed. Mean conception rates for the resulting in vitro embryos were 34.2+/-1.6% in yearling heifer recipients and 18.2+/-0.7% in lactating cow recipients. Additional oocytes (N=3312) from ovaries of anonymous donors (N unknown) collected at a commercial abattoir were fertilized using unsorted sperm, and the percentage of these that developed to blastocyst stage (20.1+/-2.9) was greater (P<0.05) than the corresponding percentage (12.2+/-2.3) achieved with sex-sorted sperm using oocytes (N=1577) from the same source. In summary, we inferred that in vitro embryo production may be a promising application of sex-sorted sperm in dairy cattle breeding, but that the biological causes of impaired embryo development in vitro and compromised conception rates of transferred embryos should be further investigated.     

Developmental capacity of bovine oocytes collected from ovaries of individual heifers and fertilized in vitro

Bovine follicular oocytes from individual heifers (n=49) were separately matured, fertilized with frozen-thawed spermatozoa and cultured with cumulus cells. Although there were great variations in the number (mean+/-SD=19.1+/-11.9) of oocytes collected from individual heifers and the percentages of the oocytes cleaved 48 hours after insemination (mean+/-SD=69.5+/-18.4) and developed to the morula stage 7 days after insemination (mean+/-SD=10.9+/-10.9), there were significant correlations between the numbers of oocytes collected and cleaved (the correlation coefficient: r= 0.9336) or developed to morula stage (r=0.6633), indicating that oocytes from different heifers have the same developmental ability after in vitro fertilization. Ten morulae and 12 blastocysts which were obtained 7 and 8 days after insemination were transferred, one by one, to each uterine horn of 11 recipients. At Day 60 of pregnancy, 8 (80%) fetuses were identified in 4 (80%) of 5 recipients into which 10 embryos were transferred at Day -1 of synchrony. However, only 3 (25%) fetuses were identified in 2 (40%) of 6 recipients into which 12 embryos were transferred at Day 0 or +1 of synchrony.     

Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP + BOEC; 3) in TALP + PHE; or 4) in TALP + BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.     

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP).Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage.Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B.Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Analysis of apoptosis in the preimplantation bovine embryo using TUNEL

The occurrence of cell death by apoptosis was examined in blastocyst and preblastocyst stage bovine embryos. Zygotes were obtained by in vitro maturation and in vitro fertilization of oocytes from abattoir derived ovaries. Two-cell to hatched blastocyst stage embryos were stained with propidium iodide to label all nuclei and by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labelling (TUNEL) to label apoptotic nuclei, and were analysed by epifluorescent and confocal microscopy. Apoptosis was first observed at the 9-16-cell stage of development, decreasing at the morula stage before increasing at the blastocyst stage. Apoptotic dead cell index in day 7 blastocysts was negatively correlated with the total number of cells; the percentage of dead cells ranged from approximately 1 to 10% and occurred predominantly within the inner cell mass. In addition, apoptotic dead cell index was significantly higher (P < 0.05) in blastocysts cultured (from the two-cell stage) in the presence of 10% fetal bovine serum compared with those developed in serum-free medium. Embryos selected for early cleavage at < 29 h after fertilization and cultured together until the blastocyst stage showed a significantly lower rate of apoptosis (P < 0.01) compared with slower cleaving embryos.     

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

In vitro production of cattle × buffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm

Interspecies hybridization of bovids occurs between domestic cattle and at least three other species; American bison (Bison bison), yak (Bos grunniens) and banteng (Bos banteng). Birth of a cattle × buffalo (Bubalus bubalis) hybrid has reportedly occurred in Russia and in China, but these reports were not authenticated. Such hybrids could be important in improving livestock production and management of diseases that impede production in tropical Africa. This study investigated hybridization between cattle and its closest African wild bovid relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce cattle × buffalo hybrid embryos in vitro, matured cattle oocytes were subjected to a standard in vitro fertilization (IVF) procedure with either homologous cattle (n = 1166 oocytes) or heterologous African buffalo (n = 1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with the homologous cattle sperm cleaved. In contrast, fertilization with buffalo sperm resulted in only a 4.6% cleavage rate. The cleavage intervals were also slower in hybrid embryos than in the IVF-derived cattle embryos. Of the cleaved homologous cattle embryos 52.2% progressed to the morula stage compared with 12.7% for the buffalo hybrid embryos. No hybrid embryos developed beyond the early morula stage, while 40.1% of the cleaved cattle × cattle embryos developed to the blastocyst stage. Transfer of buffalo hybrid IVF embryos to domestic cattle surrogates resulted in no pregnancies at 60 days post-transfer. This study indicates that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm can occur in vitro, and that a barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing the formation of hybrid embryos beyond the early morula stage. Transfer of the buffalo hybrid embryos did not rescue the embryos from development arrest.     

Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa

The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4 degrees C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5-10 microM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.