Biomedical subjects

P Lonergan

Publications and source records attributed to P Lonergan.

At least 37 records · Page 2Linked to original sources

PubMed2003

Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns.

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post-fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well-documented effects of post-fertilization culture environment on embryo gene expression and quality are highlighted.

Animals↗

PubMed2003

Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression.

We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-tau), and differentiation and implantation (LIF and LR-beta). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-beta. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-tau was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.

Animals↗

PubMed2003

Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality.

The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.

Animals↗

PubMed2003

Influence of nutrition on the effectiveness of superovulation programmes in ewes: effect on oocyte quality and post-fertilization development.

Two experiments were carried out to study the effect of nutrition on embryo development in two periods in superovulated ewes (Expt 1) and on oocyte developmental capacity during the late follicular phase (Expt 2). In Expt 1, a lower superovulation response in terms of animals ovulating (P < 0.05), ovulation rate per ewe ovulating (P = 0.1) and number of good quality embryos per animal treated (P < 0.07) was noted in ewes fed an ad libitum diet compared with ewes offered control (1.5 times the daily maintenance energy requirements, 1.5 x M) or low energy (0.5 x M) diets. Nutrition also modified the morphological and functional quality of the oocytes and embryos recovered. Thus, 92% of day 4 embryos recovered from ewes offered the control diet were classified as good embryos, compared with 70 and 82% of those recovered from ewes offered the ad libitum and low diets, respectively (P < 0.05). Ewes offered the ad libitum diet had a greater percentage of poorly developed embryos compared with ewes offered the control or low diets (P < 0.05). Ewes fed the low diet tended to have more non-fertilized oocytes than ewes offered the control diet (P = 0.09). Diet of recipient ewes to which good quality embryos were transferred on day 4 did not affect embryo quality, when assessed 12 days later (day 16 of pregnancy). However, recipient diet affected prostaglandin F(2alpha) (PGF(2alpha)) production in vitro, and uterine tissue that originated from recipient ewes on the low diet secreted more PGF(2alpha) relative to uterine tissue that originated from recipients on the control diet (P < 0.05). In Expt 2, fewer total (P < 0.05) and good quality (P < 0.01) oocytes and a lower percentage of good quality oocytes (P < 0.01) were obtained from superovulated ewes offered the ad libitum diet compared with ewes offered the low diet. In addition, cleavage rate tended to be higher (51 versus 35%, P = 0.09) in ewes offered the low diet compared with ewes offered the ad libitum diet. In conclusion, changes in diet can affect the quality of the oocyte and embryo in superovulated sheep. A lower superovulation response and a decrease in the quality of oocytes and embryos indicate that ad libitum diets are highly detrimental for superovulatory programmes when compared with low and control diets. In addition, the results from the present study indicate that a low energy diet during early embryo development increased the uterine production in vitro of PGF(2alpha) which could lead to a poor uterine environment thereby compromising the development of the embryo.

Animal Nutritional Physiological Phenomena↗

PubMed2002

Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocysts.

The aim of this study was to assess the effect of production system and of cryopreservation of ovine embryos on their viability when transferred to recipients. The experimental design was an unbalanced 2 x 2 factorial design of two embryo production systems (in vivo versus in vitro) and two embryo preservation conditions prior to transfer (transferred fresh versus transferred after vitrification/warming). For the production of blastocysts in vivo, crossbred donor ewes (n=30) were synchronised using a 13-day intravaginal progestagen pessary. Ewes received 1500 IU equine chorionic gonadotropin (eCG) 2 days before pessary withdrawal, and were mated 2 days after pessary withdrawal and embryos were recovered surgically (6 days after mating). Blastocysts were produced in vitro (IVP) using standard techniques. Recipients (n=95) were synchronised using a progestagen pessary and received 500 IU eCG at pessary removal and were randomly assigned to receive (two per recipient) in vivo fresh (n=10), in vivo vitrified (n=10), in vitro fresh (n=35) or in vitro vitrified (n=40) blastocysts. Recipients were slaughtered at day 42 of gestation and foetuses recovered. Pregnancy and embryo survival rates were recorded and analysed using CATMOD procedures. Foetal weights and crown-rump lengths were recorded and analysed using generalised linear model (GLM) procedures. There were no statistically significant interactions between the effects of embryo production system and preservation status at transfer on pregnancy rate and embryo survival. The pregnancy rate following transfer of fresh IVP blastocysts was lower (P<0.07) than that of in vivo embryos (54.3% versus 90.0%, respectively). Vitrification resulted in a decrease in pregnancy rate, the effect being more pronounced in the case of IVP embryos (54.3-5.0%, P<0.001) compared with in vivo embryos (90.0-50.0%), although the absolute change was similar (49.3% versus 40.0%). Transfer of fresh IVP blastocysts resulted in a higher proportion of single (78.9% versus 33.3%) and lower proportion of twin (21.1% versus 66.7%) pregnancies than those produced in vivo. This was reflected in a significant difference in embryo survival rate (fresh: 32.8% versus 75.0%, P<0.01; vitrified: 2.5% versus 35.0%, P<0.001, for IVP and in vivo blastocysts, respectively). Similarly, all pregnancies resulting from the transfer of vitrified/warmed IVP blastocysts were single pregnancies, while 40% of those from vitrified/warmed in vivo blastocysts were twin pregnancies; this was reflected in an embryo survival rate of 35.0% versus 75.0%, respectively. There was a significant effect (P=0.0184) of litter size on foetal weight but not on foetal length (P=0.3304). Foetuses derived from the fresh transfer of IVP blastocysts were heavier (6.4+/-0.2g versus 5.8+/-0.2g, respectively, P<0.05) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.01) than those derived from fresh in vivo blastocysts. There was no difference in these parameters as a consequence of vitrification of IVP embryos. However, in vivo blastocysts subjected to vitrification resulted in heavier (6.6+/-0.3g versus 5.8+/-0.2g, respectively, P=0.055) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.05) foetuses than their counterparts transferred fresh.

Animals↗

PubMed2002

Evolution of mRNA polyadenylation between oocyte maturation and first embryonic cleavage in cattle and its relation with developmental competence.

In this study we analyzed the pattern of polyadenylation changes that takes place between the resumption of meiosis and the first cleavage of bovine oocytes. Moreover, we investigated whether the delayed occurrence of the first cleavage division, which characterizes embryos of low developmental competence, is accompanied by an altered polyadenylation pattern of individual transcripts. We determined the polyadenylation status of a group of genes that characterize physiological processes, involved in early differentiation (Oct-4), compaction, and cavitation (beta-actin, plakophilin, connexin-32, connexin-43), energy metabolism (glucose transporter type 1, pyruvate dehydrogenase phosphatase), RNA processing (RNA poly(A) polymerase), and stress (heat shock protein 70). RNA was isolated from pools of 20 oocytes or embryos at the germinal vesicle (GV) stage, at the end of in vitro maturation, at the end of in vitro fertilization, and at the time of the first cleavage. Cleavage was assessed 27, 30, 36, 42 hr post insemination (hpi), and at the latter time the remaining uncleaved oocytes were retained as a group. Between oocyte isolation and first cleavage at 27 hpi (best quality embryos), the poly(A) tail of individual transcripts followed four patterns: no changes (beta-actin, PDP); gradual reduction (Cx-43, Oct-4, Plako); gradual elongation (Cx-32, TPA); reduction followed by elongation (PAP, HSP-70, Glut-1). If the interval between insemination and first cleavage was longer than 27 hpi (progressively lower quality embryos) further changes of polyadenylation were observed, which differed for each gene considered. These data indicated that specific changes in polyadenylation contribute to the modulation of gene expression in bovine embryos at this stage of development. Defective developmental competence is accompanied by abnormal polyadenylation levels of specific maternal mRNAs with synchrony between polyadenylation and cleavage emerging as an apparently important factor.

Animals↗

PubMed2002

Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Animals↗

PubMed2001

Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification.

The aim of this study was to assess the effect of a bovine in vitro culture system on blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were cultured in either synthetic oviduct fluid (SOF) in 5% CO2, 5% O2, 90% N2; or TCM199-granulosa cells (TCM199-GCM) in 5% CO2 in air. In vivo blastocysts were used as a control. Culture in SOF resulted in a significantly higher blastocyst yield on both Day 7 (31.3 vs 13.2%, P < 0.001) and 8 (36.8 vs 23.7%, P < 0.001) than did culture in TCM199-GCM. After vitrification, survival at 72 h of in vivo blastocysts was significantly higher than both in vitro groups, while significantly more blastocysts produced in TCM199-GCM survived compared to those produced in SOF (0, 43.5, 78.3% for SOF, TCM199-GCM and in vivo, respectively P < 0.01). In Experiment 2, SOF-GCM proved to be the best post-warming culture system of those tested and was adopted as the post-warming medium for all subsequent experiments. In Experiment 3, zygotes were cultured in SOF or SOF-GCM, in either 5% CO2 in air, or 5% CO2, 5% O2, 90% N2. In agreement with Experiment 1, culture in SOF in 5% O2 resulted in significantly more blastocysts at Day 7 (26.4 vs 17.3%, P < 0.01) and Day 8 (31.5 vs 23.2%, P < 0.01) than did culture in SOF-GCM. However, survival at 72 h post vitrification was significantly higher for SOF-GCM (44 vs 8.3%, P < 0.001). Increasing the O2 concentration to 20% significantly reduced the blastocyst eld from SOF (31.5 vs 17.3%, P < 0.001). In addition, the quality of blastocyst produced was reduced in terms of survival post vitrification (8.3 vs 0%, P < 0.05). In contrast, there was no difference in blastocyst yield (23.2 vs 25.2%) or survival (44.0 vs 36.9%) in SOF-GCM, irrespective of O2 concentration. Experiment 4 examined the duration of exposure to GCM necessary to acquire improved blastocyst quality. Zygotes were cultured in SOF; SOF until Day 3, followed by SOF-GCM for the remainder of the culture; SOF until Day 5, followed by SOF-GCM for the remainder of the culture; or SOF-GCM for the entire culture. Survival at 72 h post vitrification was significantly higher (P < 0.05) in Groups 2 (50.0%, 13/26) and 4 (55.3%, 26/47) than in Groups 1 (21.7%, 10/46) and 3 (10.8%, 4/37). In conclusion, culture system can affect blastocyst yield and quality and crytolerance is a useful indicator of blastocyst quality.

Animals↗

PubMed2001

Effect of nutrition on endocrine parameters, ovarian physiology, and oocyte and embryo development.

Reproductive efficiency in high yielding dairy cows has decreased over the past 50 years, despite significant gains in genetic selection for increased milk output. One possible reason for this decline has been a change in the nutritional intake to meet the increased energy and protein demands for higher milk production. Excess energy intake in sheep will lead to significant reductions in progesterone concentrations; the effects in cattle are not so clear. Nutrition, unless radically changed, will have little effect on gonadotropin concentrations in ruminants, and this is in contrast to the situation for pigs and for primates, where very short-term nutritional changes manifest themselves in altered gonadotropin secretion. Cattle with reduced energy intake have smaller dominant follicles and more three-wave cycles, compared with animals on higher feed intakes. One of the main areas where nutrition influences reproductive efficiency is at the level of embryo production. Several studies indicate that excess energy intake reduces the response to superovulation and also decrease the yield of embryos and alters expression of some gene constructs within the developing embryo. The mechanism of this effect is not clear but indications are that the quality of the oocytes may be compromised. Indeed recent data indicate that nutritional changes around the time of mating may have detrimental effects on the establishment of pregnancy in heifers. Thus, nutritional balancing is critical for high-yielding dairy cows, in particular. The challenge remains to modify nutritional and management strategies in such cows to maintain the levels of production made possible by genetic selection and still maintain an acceptable level of fertility.

Animal Nutritional Physiological Phenomena↗

PubMed2001

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep.

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass meal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal, for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean +/- SEM, 138.4 +/- 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean +/- SEM, 166.3 +/- 15.6, n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Animals↗

PubMed2001

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos.

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.

Animals↗

PubMed2001

Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production.

The objective of this study was to describe the ultrastructure of blastocysts derived by in vivo and in vitro methods and to investigate how the morphology is affected by exposure to cryoprotectant (10% glycerol) or cryopreservation by conventional slow freezing. In vivo derived blastocysts were characterized by a narrow perivitelline space (PvS), a continuous cover of numerous stacked microvilli (MV) on the plasma membrane, a well-defined system of cell-to-cell coupling and a large population of round or elongated mitochondria with numerous transverse cristae. Exposure of these blastocysts to cryoprotectant was manifested by shrinkage of the blastocysts and swelling of the mitochondria. Cryopreservation resulted in further shrinkage, damage to the MV, and accumulation of cellular debris. In comparison, the in vitro matured (IVM)/in vitro fertilized (IVF) in vivo cultured blastocysts displayed a wider PvS; they appeared to possess less MV and all blastocysts displayed some cellular debris in their PvS. There was also a decrease in the number of junctional contacts between the trophoblastic cells. The reaction of these blastocysts to exposure to cryoprotectant was similar to that of the in vivo derived blastocysts. However, they appeared to be more susceptible to cryopreservation. The totally in vitro produced (IVP) blastocysts displayed a wider PvS, no stacking of the MV, increased numbers of lipid droplets and a further reduction in the junctional contacts between trophoblastic cells. The IVP blastocysts sustained breakage of the zona pellucida on exposure to cryoprotectant and were extremely sensitive to cryopreservation, losing all cell structure and organization. The findings of the present study indicate that in vivo derived blastocysts possess certain structural characteristics that confer a greater tolerance on them to exposure to cryoprotectant and cryopreservation.

Animals↗

PubMed2001

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo.

The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57-78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r = 0.689, P < 0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r = 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility.

Animals↗

PubMed2001

Immunolocalization of nucleolar proteins during bovine oocyte growth, meiotic maturation, and fertilization.

During the growth phase of the bovine oocyte transcripts, polypeptides and ribosomes are accumulated in the oocyte to drive and sustain future meiotic maturation, fertilization, and early embryonic development. The oocyte also furnishes the early embryo with the components required to establish a functional transcriptionally active nucleolus at the time of maternal embryonic transition. The aim of the present study was to describe the behavior of key components of the nucleolus. The temporal localization of nucleolar proteins fibrillarin, nucleophosmin, nucleolin, RNA polymerase I (RNA pol I), topoisomerase I, upstream binding factor (UBF), and coilin 5P10 was investigated in growing and fully grown immature bovine oocytes during in vitro maturation and during the first postfertilization cell cycle using whole-mount immunocytochemistry and confocal microscopy. During the oocyte growth phase, fibrillarin, nucleophosmin, nucleolin, RNA pol I, and UBF were localized to the oocyte nucleolus. On completion of the growth phase, nucleolin and nucleophosmin appeared to migrate to the periphery of the nucleolus and into the nucleoplasm, and the proportion of oocytes displaying RNA pol I localization had decreased. Topoisomerase I was not detected at any stage. Fibrillarin appeared to be localized to large foci within the nucleolus and/or nucleoplasm. Nucleophosmin and nucleolin labeling was characterized by a homogeneous signal over the nucleolus. RNA pol I and UBF were characterized by the localization of the antibodies to individual or clustered foci in the nucleolus and/or nucleoplasm. Following oocyte nucleus breakdown (ONBD), the proteins appeared to disperse into the cytoplasm. All proteins were undetectable during meiotic maturation and were not relocalized until 5-10 h postinsemination (hpi). UBF was localized to the fertilizing sperm head of most zygotes at 5 hpi. By 10 hpi, all proteins were detected in most oocytes displaying two pronuclei. Nucleolar protein localization was exclusive to or more abundant in one pronucleus up to 20 hpi; thereafter, the pattern was more evenly distributed. Fibrillarin, nucleophosmin, nucleolin, UBF, and Pol I are present in the nuclei of growing and fully grown bovine oocytes until ONBD. They reappear at the late telophase stage of meiosis II and continue to be present up to the first mitotic division of embryo development.

Animals↗

PubMed2000

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality.

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.

Animals↗

PubMed2000

Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro.

Ram spermatozoa are most susceptible to damage during freezing between the temperatures of -10 degrees C and -25 degrees C. The objectives of the present study were to examine how freezing rate through this critical temperature zone affected the fertility of spermatozoa as assessed in vivo and in vitro. Semen from six adult rams was frozen at two different rates ("fast": 5 degrees C/min from +5 to -25 degrees C; "slow": 0.5 degrees C/min from +5 to -25 degrees C). In Experiment 1, semen from the fast and slow treatments was used to fertilize ovine oocytes that had been matured in vitro. Semen from the fast treatment yielded a higher cleavage rate (57% vs. 26%; P<0.001) and more blastocysts per oocyte (28% vs. 13%, P<0. 001) than slow-frozen. No correlation was found between fertilizing ability and viability as assessed by fluorescent probes. Experiment 2 was designed to establish the conception rates following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen as used in Experiment 1. Ewes were superovulated with FSH and inseminated by laparoscopy with frozen semen. A significant difference was found in the number of fertilized ova following embryo recovery (81.4% vs. 39.3%; P<0.001). In a further study, 119 mature cull ewes were inseminated following a 12-day synchronization treatment with frozen semen by either intrauterine (laparoscopic) or cervical insemination. Insemination with fast-frozen semen resulted in a significantly higher pregnancy rate (P<0.05) irrespective of method of insemination. The data show that freezing rate affects the proportion of spermatozoa that retain their fertilizing ability post-thawing. However, once fertilization has occurred, development to the blastocyst stage is independent of freezing rate.

Animals↗

PubMed2000

Relationship between time of first cleavage and the expression of IGF-I growth factor, its receptor, and two housekeeping genes in bovine two-cell embryos and blastocysts produced in vitro.

We have previously demonstrated that there is a clear relationship between the time interval between insemination and first cleavage in vitro and the development to the blastocyst stage of bovine embryos. In addition we have shown that this developmental ability can be linked to the stability of the mRNA for several gene transcripts measured in 2-cell bovine embryos cleaving at different times. The aim of this study was to examine the relationship between bovine embryo developmental competence, assessed in terms of time of first cleavage, and the expression of insulin-like growth factor-I (IGF-I) ligand and receptor, hypoxanthine phosphoribosyl transferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD). The expression of beta-actin was used as a reference value. No differences were observed in the mRNA expression of G6PD and HPRT genes between male and female 2-cell embryos. However, the expression of these two genes was significantly higher in female blastocysts than in male blastocysts. Moreover, when the relative amount of G6PD and HPRT mRNA detected in these groups of male and female embryos was compared, there was a significant relationship between the time of first cleavage and the relative amount of mRNA: 2-cell embryos and blastocysts derived from oocytes that cleaved at 27 and 30 hr post insemination had higher levels of mRNA for G6PD and HPRT than those that cleaved after 33 hr. IGF-I ligand and receptor was detected in all blastocysts analyzed, irrespective of stage of development or time of first cleavage. In addition, the receptor was detected in all 2-cell embryos examined. In contrast, while IGF-I ligand was found in all 2-cell embryos that cleaved at 27 and 30 hpi, it was only found in some of those cleaving between 33 and 36 hpi and in none of those cleaving after 36 hr. In conclusion, we have demonstrated differences in gene expression in the early embryo that are reflective of differences in developmental competence between early- and late-cleaving zygotes.

Animals↗

PubMed2000

Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I.

In this study we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, inhibits meiotic resumption in bovine oocytes by blocking germinal vesicle breakdown in a dose-dependent manner. A concentration 100 microM blocked over 60% of oocytes, while 150 microM inhibited almost all oocytes compared to the control in which over 80% resumed meiosis. Following a second 24 hr culture under conditions permissive to normal maturation, almost all (95%) of blocked oocytes resumed meiosis and progressed to metaphase II. In terms of developmental competence, oocytes maintained in meiotic arrest for 24 hr with 100 microM exhibited a similar capacity to develop to the blastocyst stage as nonblocked control oocytes following maturation, fertilization, and culture in vitro. Cryopreservation was employed as a tool to detect differences in the oocyte viability between blocked and control oocytes. Cleavage of oocytes was significantly reduced following vitrification and activation both in BL-I treated (40.2% vs. 71.9%, P < 0.05) and the control groups (45.6% vs. 81.7%, P < 0.05). However, BL-I treated oocytes were less likely to develop into blastocysts following vitrification (20.0% from vitrified vs 42.5% from nonvitrified cleaved oocytes, P < 0.05, based on cleaved oocytes) compared to nontreated oocytes (34.0% from vitrified vs. 42. 9% from nonvitrified oocytes, P < 0.05). These results demonstrate the feasibility of maintaining bovine oocytes in artificial meiotic arrest without compromising their subsequent developmental competence and may represent a tool for improving the development of less competent oocytes.

4-Butyrolactone↗