Relationship between Oxygen Consumption and Sex of Bovine In Vitro Fertilized Embryos

The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.     

Ultra‐Structural Alterations in In Vitro Produced Four‐Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four‐cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow‐freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra‐structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.     

几种冷冻条件对牛体外受精卵发育率的影响

试验比较了乙二醇 (EG)、丙二醇 (PG)、二甲基亚砜 (DMSO)、甘油 (G)和不同的投液氮温度 (- 33℃或 - 4 0℃ )对牛体外受精卵冷冻后发育率的影响。结果 :投液氮温度无论是 - 33℃或 - 4 0℃ ,均以 1.5 mol/ L EG的冷冻效果为最好 ,与 1.5 mol/ L PG相比 ,受精卵的发育率差异显著 (39.7% vs19.2 % ;4 7.8% vs2 4 .7% ,P <0 .0 5 ) ;同 1.5mol/ L DMSO和 1.5 mol/ L G相比 ,受精卵的发育率差异极显著 (39.7% vs 16 .1% or 13.3% ;4 7.8% vs 19.2 % or17.3% ,P <0 .0 1)。 - 4 0℃投液氮 ,受精卵冷冻后的发育率略高于 - 33℃ ,但差异不显著 (P >0 .0 5 )。以 2 5 % EG 2 5 % PG作为细胞外玻璃化溶液对牛体外受精卵进行冷冻 ,冷冻后受精卵的发育率达 5 8.9% ,高于用 1.5 mol/ L EG冷冻在 - 4 0℃投液氮这一处理 (47.8% ) ,但无统计学上差异 (P >0 .0 5 )。然而 ,试验组冷冻后受精卵的发育率均极显著低于对照组 (80 .6 8% ,P <0 .0 1)。结果表明 ,投液氮温度以 - 4 0℃为较好 ,防冻剂以 EG的冷冻效果为最佳。玻璃化冷冻法完全可以应用于冷冻牛体外受精卵     

体外生产的牛胚胎超微结构研究

利用两种常规体外培养系统研究了体外培养及不同培养条件对牛体外受精胚胎早期发育各时期超微结构的影响。结果显示:体外发育的牛胚胎在发育的各个时期(从原核期到囊胚)胞质中均含有丰富的脂滴,胚胎中特别是在桑椹胚及囊胚阶段可见到一些异常细胞。两系统下发育的胚胎中脂滴形态不同,从桑椹胚阶段开始,胚胎线粒体的发育在M199 BOEC FCS培养系统中明显滞后于其在SOFaa BSA培养系统中的对照。     

Bovine Immunodeficiency Virus in Relation to Embryos Fertilized In Vitro

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.     

Lipid Droplet Analysis Using In Vitro Bovine Oocytes and Embryos

The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post‐mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM‐199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi‐fine toluidine blue‐stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro‐cultured embryos.     

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 μl of BoHV-1, Los Angeles strain 10^7.5 TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.     

Vitrification of Bovine Blastocysts Produced In Vitro Inflicts Selective Damage to the Inner Cell Mass

In contrast to the embryos derived from live animals, the embryos produced in vitro undergo increased damage and reduced survival after cryopreservation, particularly when produced with serum. In medium containing serum, retinoic acid increases cell numbers in the inner cell mass and the trophectoderm without altering their relative proportions in the bovine blastocyst. In this work, in medium without serum, we analyzed the contribution of retinoic acid to the development of blastocyst and survival to vitrification, and found a strong cell reduction in the inner mass when compared to the trophectoderm. Day-6 in vitro -produced morulae were treated for 24 h with retinoic acid (0.7 and 1.4 μ m ) and subsequently cultured without additives for a further 24 h period. Day-8 blastocyst production and cell counts in hatched blastocysts were unaffected by retinoic acid. However, Day-7 expanded, vitrified embryos produced with retinoic acid 1.4 μ m survived at lower rates than controls when cultured after warming. Vitrification greatly reduced cell numbers in the inner mass (p < 0.0001), while cells in the trophectoderm remained unaltered. Differential cell counts analysis in blastocysts should be taken up to replace unspecific determination of total cells to appreciate substantial modifications in their exact terms. The strong reduction we found in the inner cell mass could explain why in vitro survival to cryopreservation is sometimes scarcely informative on the viability of the embryo after transfer to recipients.     

Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in Vitro

Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1-15%, (3) 16-30%, (4) 31-50%, (5) 51-75% and (6) 76-100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16-50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1-15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage.     

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

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

Developmental Capabilities of Embryos Produced In Vitro from Prepubertal Lamb Oocytes

Breeding from prepubertal females, known as juvenile in vitro embryo transfer (JIVET), reduces the generation interval and increases the rate of genetic gain in animal breeding programs. While the birth of the first lambs from prepubertal ewes occurred nearly 30 years ago; and there is considerable interest in the commercialization of this technology, its efficiency remains too low. The advent of in vitro production (IVP) of embryo resulted in the more widespread use of JIVET. Morphologic and metabolic differences coupled with reduced in vitro and in vivo development of oocytes derived from prepubertal animals have been reported. Research has been undertaken to optimize donor selection and hormone stimulation methods in an attempt to reduce the variability and increase the proportion of donors responding to hormone stimulation and increase oocyte developmental competence. Yet, this variation persists and the development of oocytes and embryos from prepubertal animals remains reduced when compared with adults. Recent improvements to JIVET, resulting from a modified hormone stimulation regime, have eliminated the failure of donors to respond to hormone stimulation, and increased both the number and developmental competence of oocytes harvested from very young prepubertal lambs. This increased efficiency has facilitated the incorporation of other reproductive technologies such as sperm sexing with JIVET, resulting in the birth of lambs of a pre-determined sex from prepubertal lambs. Increased rates of genetic gain in sheep breeding programs can be achieved by combining sexed sperm with oocytes obtained from lambs as young as 3–4 weeks of age. Continued increases in the efficiency of JIVET resulting from further improvements to hormone stimulation regimes and an increased understanding of the differences between oocytes from adult and prepubertal animals will result in the commercialization of this technology.     

Production of Middle White Piglets after Transfer of Embryos Produced In Vitro

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing Middle White piglets. After collection from three retired Middle White sows, a total of 222 oocytes were matured, fertilized and cultured in vitro, and a total of 50 embryos from the 4-cell to blastocyst stage were produced by the 4th or 5th day. These embryos were transferred individually into three recipients along with 5 in vivo-derived Duroc blastocysts. All of the recipients became pregnant, and they farrowed a total of 9 Middle White and 9 Duroc piglets. These results suggest that in vitro embryo production using ovaries from retired sows is useful for reproduction of pigs of pure breeds including the Middle White for breeding activities and conservation/utilization of genetic resources.     

Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming.     

In Vitro Development of Bovine Embryos after Fertilization using Semen from Different Donors

Contents: The aim of this study was to determine whether the semen donor and/or heparin concentration influences the rate of fertilization of bovine follicular oocytes and their subsequent embryonic development in vitro. Frozen-thawed semen from five highly fertile bulls was treated with one of four concentrations of heparin (0.5,1.0, 2.0 and 5.0 μg/ml) on a 5 x4 factorial basis in an IVM-NF programme. Zygotes/oocytes were cultured in frozen-thawed bovine oviduct cell-conditioned medium for 6 days. The use of semen from different bulls resulted in significantly (P < 0.001) different rates offertilization, as judged by cleavage rates of the oocytes at 72 h post insemination, and subsequent embryonic development through the'8-cell-block'(P < 0.05) in vitro. Development up to the morula/blastocyst stage, however, did not differ significantly (P = 0.06) among groups of oocytes fertilized with spermatozoa from different bulls. Heparin levels ranging from 0.5 to 5.0 μg/ml did not differ in their effect on in vitro fertilization as judged by the rate of normally cleaved oocytes (P = 0.14). The overall parthenogenetic division rate at 72 h post insemination was 12.4% and was not influenced by the heparin concentration. There was a linear relationship (P < 0.001) between fertility estimates based on AI and the estimates basedon the first cleavage following in vitro fertilization.     

In Vitro Development of Bison Embryos Using Interspecies Somatic Cell Nuclear Transfer

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non‐domestic animal species. However, problems arise during the development of these embryos, which may be related to species‐specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria‐related genes NRF1, MT‐CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT‐CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.     

Comparison of Two Semen Extenders in Terms of In Vitro Development of Bovine Embryos Following IVF

In order to conform with current EC standards with regard to antibiotic cover, the Norwegian Cattle Association is currently investigating the use of Biladyl® as an alternative to the milk-based extender which has been traditionally used in Norway. A study was carried out to investigate the effect of using semen frozen with either milk extender or Biladyl® on the outcome of in vitro fertilization and embryo culture. Semen from 6 Norwegian Red bulls was used. There was a significant difference p<0.05 in terms of cleavage rate between the 2 extenders for 1 bull, 78.2% vs 94.9% for milk and Biladyl® extenders, respectively, and for the overall total of 71.3% vs 76.1% for milk and Biladyl® extenders, respectively. There were no significant differences in terms of blastocyst yield amongst any of the bulls. In conclusion, the results suggest that Biladyl® can be used as a replacement for the traditional milk-based extender without any adverse effects on blastocyst yields following in vitro fertilization.     

Apoptosis-independent Poor Morphology of Bovine Embryos Produced by Multiple Ovulation

In multiple ovulation and embryo transfer (MOET) programmes in cattle, a considerable number of morphologically poor-quality embryos continue to be produced; this is one of the limiting factors of the technique. Apoptosis has often been implicated in developmental arrest and fragmentation; these are regarded as poor traits of embryonic quality in mammalian pre-implantation embryos. In the present study, apoptosis was assessed in morphologically poor-quality embryos in comparison with good-quality embryos that were recovered from a MOET programme. Retarded embryos (two to 16 cell stage), morulae with severe fragmentation and morphologically good-quality morulae recovered from superstimulated cows at day 7 post-insemination were subjected to TdT-mediated dUTP nick-end labelling (TUNEL) and Hoechst staining. Cell nuclei that showed both TUNEL staining and apoptotic morphology were considered to be apoptotic. Apoptotic index (AI) was calculated as the percentage of apoptotic cells per embryo. Fifteen of 17 retarded embryos and 10 of 15 morphologically poor-quality morulae did not show signs of apoptosis. The mean AIs in the morphologically poor-quality embryos (two to 16 cell stage, 2.2%; poor morulae, 1.3%) were as low as that in the good-quality embryos (2.9%). These results suggest that another mode of developmental arrest and/or fragmentation that is independent of apoptosis occurs in morphologically poor-quality embryos recovered from MOET programmes.