Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

The effect of season on oocyte quality and developmental competence in Italian Mediterranean buffaloes (Bubalus bubalis)

At Italian latitudes, buffalo (Bubalus bubalis) is a seasonally polyestrous species, showing an improved reproductive efficiency when daylight decreases (autumn). The aim of the present study was to evaluate the influence of the season on buffalo oocyte recovery rate, on oocyte quality, assessed on morphological basis, and developmental competence after in vitro fertilization. For this purpose, buffalo ovaries were collected from a local abattoir and the oocytes obtained by aspirating the follicles were evaluated, classified and, if considered of good quality, devolved to the different procedures of IVEP. In general, no differences were found in terms of oocyte recovery per ovary among seasons, but interestingly, the percentage of small oocytes was higher (P<0.05) during spring and summer (0.9±0.1 and 0.9±0.2) compared to autumn and winter (0.3±0.1 and 0.2±0.1). Both cleavage and embryo rate increased during the period from October to December (71.7±3.1 and 26.5±2.1, respectively) compared to the period from April to June (58.0±2.4 and 18.8±1.6, respectively), thus reflecting the in vivo reproductive behavior. Nevertheless, it is worth emphasizing that transferrable embryos were produced in vitro, even during the unfavorable season, but with decreased efficiency. In conclusion, these results suggest to avoid the oocyte collection during spring when planning OPU trials in order to save resources and improve the benefits/costs ratio.     

Breed influences on in vitro development of abattoir-derived bovine oocytes

ABSTRACT: BACKGROUND: There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development. METHODS: The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds. Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined. RESULTS: Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor's breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef. CONCLUSIONS: The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.     

Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal and adult somatic cell nuclei into bovine (Bos indicus) oocyte cytoplasm and their subsequent development

Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal fibroblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal fibroblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal fibroblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a significantly higher cleavage rate (P < 0.05) for fused nuclear transferred embryos produced by fetal fibroblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal fibroblasts was higher (P < 0.05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts confirmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells.     

Effect of age,pregnancy, and tuberculosis status on oocyte parameters in African buffalo

In the past decades, the African buffalo (Syncerus caffer) population has steadily declined; an estimated reduction of 31.2% over 3 generations has resulted in classifying it as near threatened by the International Union for Conservation of Nature. Therefore, the aim of this work was a preliminary evaluation of the size and number of ovarian follicles and oocyte quality and quantity in African buffaloes in relationship to age, tuberculosis, and pregnancy status to assess feasibility of in vitro embryo production (IVEP) as a tool for conservation and propagation of this species. The study occurred during the winter dry season (Jul–Aug) of 2018 within Huluhluwe-Imfolozi National Park in Kwa-Zulu Natal, South Africa during the routine culling procedures of African buffalo carried out at the park by the provincial authorities as means of disease management and population control. We obtained ovaries from 39 adults and 10 juveniles, and transported them to the field laboratory where we counted follicles, collected oocytes by aspiration and slicing, and classified oocytes according to their morphology. Adult animals had more small and medium follicles compared to juveniles (small: 14.5 ± 0.1 [SE] vs. 14.0 ± 0.1; medium: 4.6 ± 0.7 vs. 2.0 ± 1.3); however, juvenile animals had more cumulus-oocyte complexes (COCs). Pregnancy and tuberculosis status did not affect COC recovery rate and quality. The oocyte recovery rate is comparable to cattle and higher than in water buffalo (Bubalus bubalis). Therefore, our results suggest that IVEP could be an effective tool for conservation of valuable germplasm in African buffalo. The use of aspiration, using an 18-gauge needle and syringe, followed by slicing the ovary with a scalpel blade increased the recovery of suitable COCs (aspiration = 37.2 ± 3.02%; aspiration and slicing = 46.1 ± 3.02%), supporting the use of aspiration and slicing to optimize oocyte collection for conservation of African buffalo.     

Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows

The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti‐mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides, <10% of oocytes (with CC biopsies) derived from dairy cattle were able to develop to the blastocyst stage. In addition, a higher level of expression and a positive correlation were observed in the expression of most of the genes evaluated (LUM, KRT18, KRT8, CLIC3, BMPR1B, and SLC38A3) in the cumulus–oocyte complexes that produced blastocysts versus those which did not develop correctly (arrested development). Further, use of BMP15 in the maturation of oocytes from dairy cattle seems to increase competence, modulating the expression of OCT4, SOX2, CDX2, GATA6, and TP1 in resulting blastocysts.     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.     

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.     

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.     

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.     

Effect of cysteamine during in vitro maturation on buffalo embryo development

The purpose of this study was to evaluate whether the addition of cysteamine during in vitro maturation (IVM) of buffalo oocytes enhances embryo development. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were matured in vitro in TCM 199 supplemented with 10% fetal calf serum (FCS), 0.5 microg mL(-1) FSH, 5 microg mL(-1) LH, 1 microg mL(-1) 17 beta estradiol and 0 (control), 50, 100 or 200 micromol L(-1) of cysteamine for 24 hours. The matured oocytes then were fertilized and cultured for 7 days. No beneficial effect on maturation and cleavage rate was related to the addition of cysteamine. However, the percentage of embryos that developed to compact morula and blastocyst stage was significantly higher (P < or = 0.01) for oocytes matured in medium containing 50 micromol L(-1) of cysteamine than it was for oocytes matured with 0, 100 and 200 micromol L(-1) cysteamine (22.6% vs 14.9%, 15.7% and 13% respectively); moreover, the addition of 50 micromol L(-1) of cysteamine during IVM significantly (P < or = 0.01) increased the proportion of transferable quality (Grades 1 and 2) embryos (19.3% vs 11.3%, 11.6% and 11.2% respectively). The present study showed that adding a thiol compound (such as cysteamine) to the IVM medium improves buffalo in vitro embryo production (IVEP) efficiency, which so far has been unsatisfactory.     

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.     

In vitro developmental competence of buffalo oocytes collected at various stages of the estrous cycle

The objective was to determine the in vitro developmental competence of buffalo oocytes collected from abattoir-derived ovaries at various stages of the estrous cycle and follicular status. In Experiment 1, ovaries (n=476 pairs) were collected and divided into the following five groups: (a) ovaries with a corpus hemorragicum and no dominant follicle (CH-NO-DF); (b) ovaries with a mature functional corpus luteum (CL) and a dominant follicle (CL-DF); (c) ovaries with a mature functional CL and no dominant follicle (CL-NO-DF); (d) ovaries with a regressing CL and a dominant follicle (RCL-DF); and (e) ovaries without any luteal structures and only small follicles (ANEST). In Experiment 2, 144 pairs of ovaries with a CL (or regressing CL) and a dominant follicle were collected and follicles were classified as dominant, largest subordinate, and subordinate. In both experiments, the dominant follicle was defined as any follicle >10mm in diameter that exceeded the diameter of all other (subordinate) follicles. Although oocytes were collected from each group of ovaries, only Grades A or B oocytes were used for in vitro embryo production. Cleavage rates were higher (P<0.05) from oocytes collected from ovaries in the CH-NO-DF (59.6%) and CL-NO-DF (59.2%) groups than those collected from CL-DF (52.2%) and ANEST (43.6%) groups. The yield of transferable embryos was higher (P<0.05) from oocytes collected from CH-NO-DF (27.4%) and CL-NO-DF (24.0%) ovaries than from CL-DF (16.2%), RCL-DF (15.4%), and lowest (P<0.05) from ANEST (8.8%). In Experiment 2, oocytes from the dominant follicle had a higher (P<0.05) cleavage rate (65.2 %) and transferable embryo yield (30.2%) than those collected from the largest subordinate and subordinate follicles. In conclusion, oocyte competence depended on the morphofunctional state of ovaries. Oocyte development was maximal in pairs of ovaries with a corpus hemorragicum or CL and no dominant follicle; in paired ovaries with a CL and a dominant follicle, development was maximal in oocytes derived from the dominant follicle.     

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus)

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.     

The effect of vitamins during maturation of caprine oocytes on subsequent developmental potential in vitro

Only a small proportion of goat oocytes selected for in vitro oocyte maturation (IVM) can successfully complete cytoplasmic maturation and support embryonic development. To produce goat blastocysts more efficiently in vitro, it is necessary to identify factors required during oocyte maturation. The objective of this study was to determine the role of vitamins during maturation of caprine oocytes in semi-defined medium on subsequent developmental capacity in vitro. Cumulus oocyte complexes (COCs) collected from a local abattoir were matured in synthetic oviductal fluid (SOF) medium supplemented with BSA, LH, FSH, and EGF in the presence or absence of MEM vitamins for 24 h. The COCs were co-incubated with frozen-thawed sperm in Bracket and Oliphant fertilization medium for 18-22 h. Embryos were cultured in G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 72 h. Addition of vitamins significantly increased (P<0.05) overall blastocyst development (16.4+/-1.2% versus 12.3+/-1.1%), and tended to increase (P<0.06) the percentage of cleaved embryos (61.4+/-3.0% versus 52.7+/-2.6%). Addition of MEM vitamins to SOF maturation medium significantly increased (P<0.05) mean blastocyst cell number compared with control medium (107.7+/-6.0 versus 85.1+/-6.3). Hatched blastocysts tended to have increased (P<0.06) cell numbers in the vitamin-treated group (150.5+/-8.4 versus 123.4+/-8.8). These results suggest that addition of vitamins during oocyte maturation is beneficial for subsequent blastocyst development and viability.     

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.     

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.