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Bovine in vitro fertilisation technology has been widely exploited in commercial settings. The majority of in vitro -derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of in vitro -derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if in vitro fertilisation technology is to be adopted in the field. Cryopreservation is the process of cooling and storing living cells at very low temperatures to maintain their viability after warming. Vitrification is a popular way to freeze 'ice-free' human oocytes and embryos. This review discusses how vitrification is being optimised and standardised for in vitro -derived cattle embryos of defined quality as a useful tool for bovine IVF in commercial settings. [ABSTRACT FROM AUTHOR]

Advancing the age at which puberty and subsequent sexual maturation is attained in cattle is central to the reproductive and economic efficiency of both beef and dairy production systems worldwide. Onset of puberty in both male and female cattle is regulated by a complex network of biochemical processes and involves interaction among many key metabolic, neuroendocrine and reproductive tissues. Although our understanding of the biochemical interplay that conditions and eventually triggers the pubertal process has improved in recent years, much of the intricate mechanistic detail still eludes us. Environmental factors, such as nutritional management, as well as the genetic makeup of the animal undoubtedly affect the timing of puberty in cattle. In particular, there is now overwhelming evidence to support the importance of early life nutrition in regulating the timing of puberty in both bulls and heifers. For both genders, there is significant evidence that an improved metabolic status, early in calfhood, advances maturation of the hypothalamic- pituitary-gonadal axis, therefore facilitating earlier sexual development. Although advancing sexual maturation is a desirable goal, it is important that any strategy used does not impinge upon normal gametogenesis or postpubertal fertility potential. To this end, the aim of this review is to discuss the underlying biology of puberty in cattle with particular emphasis on the role of nutritional management during early calfhood in: (1) advancing the maturity of the hypothalamic- pituitary-gonadal axis; and (2) implications for the quality of gametes and subsequent fertility. [ABSTRACT FROM AUTHOR]

The article presents abstracts on embryo culture which include transcription profiling in oocytes-embryo and granulosa cells from bovine, effect of bovine oviductal fluid on DNA methylation of bovine blastocsts produced in vitro, and bone morphogenetic protein signaling during interaction of the bovine embryo with oviductal epithelial cells in vitro.

In the field of ‘single cell analysis’, many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na]+ (m/z 725) and phosphatidylcholine [PC (32:0)+Na]+ (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development. [ABSTRACT FROM AUTHOR]

The aim was to examine the effect of a single intramuscular (i.m.) injection of equine chorionic gonadotrophin (eCG) on Day 3 after oestrus on corpus luteum (CL) development, circulating progesterone and conceptus development in cross-bred beef heifers. In Experiment 1, heifers received: (1) saline, or a single i.m. injection of eCG on Day 3 at (2) 250IU (3) 500IU (4) 750IU or (5) 1000IU. Administration of eCG resulted in increased luteal tissue area and progesterone and oestradiol concentrations compared with controls. In Experiment 2, heifers received (1) a progesterone-releasing intravaginal device (PRID Delta) from Day 3 to 5 or (2) a PRID Delta from Day 3 to 5 plus a single injection of 750IU eCG on Day 3. In vitro-produced blastocysts (n=10 per recipient) were transferred on Day 7 and heifers were slaughtered on Day 14 to assess conceptus development. Administration of eCG reduced the number of short cycles (6.3% vs 31.3%) and increased mean luteal tissue weight (P=0.02). Insertion of a PRID Delta on Day 3 resulted in an elevation (P<0.05) in serum progesterone until removal on Day 5. Administration of eCG at the time of PRID Delta insertion resulted in higher progesterone levels (P<0.05) from Day 10 onwards. Conceptus dimensions were not affected. In conclusion, a single injection of eCG on Day 3 increased CL size and progesterone concentrations and, when given in conjunction with a progesterone-releasing device, appeared to reduce the number of short cycles, presumably due to its luteotrophic nature. The implications of the elevated oestradiol concentrations for embryo quality require further study. [ABSTRACT FROM AUTHOR]

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3 mM VPA for 24 h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage. Histone deacetylase inhibitors (HDACis) have been widely used to improve the developmental competence of embryos derived from somatic cell nuclear transfer (SCNT). The present study was conducted to elucidate the effects of valproic acid (VPA), a recently-characterized HDACi, on the early development of bovine SCNT embryos and the underlying mechanisms of these effects. We revealed that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage. [ABSTRACT FROM AUTHOR]

Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36 h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos. [ABSTRACT FROM AUTHOR]

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus. Little is known about the molecular mechanisms that regulate bovine trophoblast development. In current study, we demonstrated that impaired expression of two key regulatory proteins, E-cadherin and β-catenin along with defective β-catenin signaling activation occurs in trophoblast derived from bovine somatic nuclear transfer (SCNT). These deficiencies during embryo attachment could explain the insufficient placentation in the SCNT-derived fetus. [ABSTRACT FROM AUTHOR]

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24 072 probe sets representing over 23 000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24 -- 35 months old) versus prepubertal (9 -- 10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.4%) and 490 (2.0%) genes were differentially expressed in prepubertal and adult bovine in 8- to 16-cell and blastocyst stage embryos, respectively. Out of these, 218 and 248 genes were upregulated, while 373 and 242 were downregulated in prepubertal and adult 8- to 16-cell and blastocysts stage embryos, respectively. Gene ontology classification regarding biological process, molecular functions and cellular component revealed diversity in transcript abundances between prepubertal and adult groups in both the distinct developmental stages. Quantitative reverse transcription -- PCR validated the expression differences of some selected transcripts as identified by microarray analysis. To our knowledge, this is the first report indicating the significant number of genes differentially expression (>2-fold, P < 0.01) in preimplantition embryos between adult and prepubertal Japanese Black cattle during in vitro development. The development competence of oocytes derived from prepubertal animal is compromised as compared to their adult counterparts. The aim of the present study was to identify gene expression profile difference in embryos derived from adult and prepubertal Japanese Black cattle. The identification of differences in transcripts in the prepubertal and adult embryos could represent a valuable tool for the improvement of protocols for the manipulation of prepubertal oocytes, increasing the efficiency in the utilisation of such oocytes. [ABSTRACT FROM AUTHOR]

Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts and that this may be related to differences in glucose metabolism. In addition, an inhibition of phosphatidylinositol 3-kinase (PI3-K) inhibits glucose uptake in murine blastocysts. Therefore, the aim of this study was to identify and compare the expression of proteins involved in glucose metabolism (hexokinase-I, HK-I; phosphofructokinase-1, PFK-1; pyruvate kinase1/2, PK1/2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; glucose transporter-1, GLUT-1; and glycogen synthase kinase-3, GSK-3) in male and female bovine blastocysts to determine whether PI3-K has a role in the regulation of the expression of these proteins. Hexokinase-I, PFK-1, PK1/2, GAPDH and GLUT-1 were present in bovine embryos. Protein expression of these proteins and GSK-3 was significantly higher in male compared with female blastocysts. Inhibition of PI3-K with LY294002 significantly decreased the expression of HK-I, PFK-1, GAPDH, GSK-3 A/B and GLUT-1. Results showed that the expression of glycolytic proteins HK-I, PFK-1, GAPDH and PK1/2, and the transporters GLUT-1 and GSK-3 is regulated by PI3-K in bovine blastocysts. Moreover, the differential protein expression observed between male and female blastocysts might explain the faster developmental kinetics seen in males, as the expression of main proteins involved in glycolysis and glycogenogenesis was significantly higher in male than female bovine embryos and also could explain the sensitivity of male embryos to a high concentration of glucose, as a positive correlation between GLUT-1 expression and glucose uptake in embryos has been demonstrated. This study examined the protein expression of enzymes involved in the glycolytic and glycogen pathways in bovine embryos. Results demonstrate that four important glycolytic proteins (HK-I, PFK-1, GAPDH and PKM1/2), the protein transporter GLUT-1 and the glycogenogenetic GSK-3 protein are expressed in bovine embryos as components of glucose energetic metabolism and that the protein expression is critically regulated by PI3-K. Moreover, protein expression of HK-I, PFK-1, GAPDH and PKM1/2, and GSK-3A and GSK-3B phosphorylation is different in male and female bovine embryos, which may be related to the differences in the kinetics of development found between male and female bovine embryos and also could explain the sensitivity of male embryos to high concentrations of glucose. [ABSTRACT FROM AUTHOR]

This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100 μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P < 0.001) whereas inclusion of Trolox had no effect (P > 0.05). Whereas lipoproteins reduced zygote development to blastocysts (P = 0.03), Trolox facilitated increased development (P < 0.001) and counteracted the reduction observed with lipoproteins (interaction, P = 0.009). Lipoproteins also compromised (P < 0.001) but presence of Trolox (P > 0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P < 0.001). Liver weight (g kg -- 1 fetal weight) was greater (P = 0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P = 0.002); superior embryo grades were also associated with increased numbers of placentomes (P = 0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70. Bovine embryo quality after in vitro culture is an important determinant of subsequent developmental potential after transfer to recipient animals. We found for the first time that the negative effects of lipoprotein-inclusion and positive effects of antioxidant-inclusion during in vitro culture were reflected in subsequent fetal development. The results emphasise the important of optimising specific culture conditions for subsequent embryo development. [ABSTRACT FROM AUTHOR]

The objectives of the present experiment were to determine whether supplementation with progesterone (LO, 1ngmL-1or HI, 100ngmL-1) during either the first (Culture-1, Day 1 to 3) or second (Culture-2, Day 4 to 7) phase of culture of in vitro-produced embryos alters embryo development, embryo metabolism or blastocyst cell number. The percentage of oocytes that cleaved, the percentage of cleaved embryos that developed to the morula stage or greater, the blastocyst stage or greater or the hatched blastocyst stage were similar among treatments. Quantities of glucose metabolised per blastocyst per hour were similar, but when metabolic data was normalised for numbers of cells in each blastocyst, the LO treatment during Culture-2 metabolised more glucose (P=0.03) compared with all other treatments. Embryos receiving LO progesterone tended to have greater (P=0.085) metabolism of glucose compared with embryos receiving HI progesterone. Quantities of pyruvate oxidised per blastocyst per hour, and per cell, were similar among treatments. The number of cells per blastocyst in the control group was increased (P=0.039) compared with cells in progesterone-treated groups. In conclusion, supplementation with progesterone during the culture of in vitro-produced embryos does not appear to improve embryo characteristics. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the incidence of chromosomal abnormalities in bovine blastocysts produced by IVF with unsorted, X-sorted or Y-sorted spermatozoa. In Experiment 1, individual blastocysts were processed to examine the incidence of mixoploidy using fluorescent in situhybridisation. Overall, 80% (44/55) of blastocysts were mixoploid (10/15, 14/15 and 20/25 for X-sorted, Y-sorted and unsorted spermatozoa, respectively; P>0.05). However, the prevalence of abnormal XY chromosome complements was relatively low in all groups; on average, only a small fraction of the total nuclei per embryo appeared polyploid (1.64%, 5.62% and 6.0% for X-sorted, Y-sorted and unsorted spermatozoa, respectively). Interestingly, 20% (5/25) of blastocysts derived from unsorted spermatozoa were found to be chimeric (XX/XY). In Experiment 2, chimeric embryos were detected among the blastocysts derived from two of five sires tested. In addition, one chimeric blastocyst was detected among nine in vivo-derived blastocysts obtained following AI. In conclusion, based on the results of the present study, the incidence of chromosomal abnormalities did not different between blastocysts derived from sex-sorted or unsorted spermatozoa. In addition, the occurrence of mixed sex chimeras was not limited to a single sire and was not unique to blastocysts derived from IVF. [ABSTRACT FROM AUTHOR]

In cattle, the embryo production rate after superovulation varies between individuals and is difficult to predict. Recently, we proposed that anti-Müllerian hormone (AMH) plasma levels measured before treatment can help predict superovulatory responses. To establish whether blood measurement of AMH can help predict the number of embryos produced by a given cow after superovulation, data collected over 4 years from 45 dairy cows submitted to repeated embryo production were analysed in a retrospective study. A high within-animal repeatability (0.38 and 0.36) and a strong effect of the father of the donor cow (P< 0.01) were observed for the numbers of collected and transferable embryos, respectively. AMH concentration, measured in the plasma of donor cows during first lactation and several months before the start of the embryo production campaigns, was found to be highly correlated with the maximal number of collected (P< 0.0001) and transferable (P< 0.01) embryos per cow. In conclusion, the capacity of embryo production is a repeatable and probably heritable trait in the cow, and blood measurement of AMH in potential donor cows could be of value in determining a cow's intrinsic capacity to produce transferable embryos. [ABSTRACT FROM AUTHOR]

Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of development to survive the vitrification procedure using cryotop devices. Day 7 and Day 8 embryos were classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate of vitrified–warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment, vitrified–warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, results for cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8 expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts. When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded and hatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowest survival rates were recorded for non-expanded blastocysts (26%–54%) compared with the other developmental stages, particularly those vitrified at Day 8 (≤40%). The DNA integrity index obtained after vitrification–warming was comparable to that for control fresh blastocysts, regardless of the length of embryo culture, the developmental stage of the embryo or the source of the oocytes. Our findings suggest that the cryotop vitrification method is particularly useful for the cryopreservation of blastocysts presenting with a high degree of expansion (expanded or hatched blastocysts), particularly when vitrification is performed after 7 days of in vitroembryo culture. [ABSTRACT FROM AUTHOR]

Although the transfer of embryos is much less likely to result in disease transmission than the transport of live animals, the sanitary risks associated with embryo transfer continue to be the subject of both scientific investigations and adaptations of national and international legislation. Therefore, the implications are important for veterinary practitioners and livestock breeders. In vivo-derived and in vitro-produced embryos are widely used in cattle and embryos from other species, such as sheep, goats, pigs and horses, are also currently being transferred in fairly significant numbers. Bearing in mind the wide variety of embryos of different species and the correspondingly large number of viruses that are of concern, it is expedient at this time to look again at the importance of the zona pellucida (ZP) as a barrier against viruses and at the susceptibility or otherwise of embryonic cells to viral infection if ever they are exposed. For embryos with an intact ZP, viral infection of the embryo is unlikely to occur. However, the virus may stick to the ZP and, in this case, International Embryo Transfer Society (IETS) washing procedures in combination with trypsin treatment are mandatory. A caveat is the fact that currently more and more types of embryos are becoming available for transfer and scientific data cannot be extrapolated from one species to another. These topics are discussed in the present review. [ABSTRACT FROM AUTHOR]

Embryo transfer is a biotechnology that has been used worldwide to increase the production of offspring from female bovines. Treatments to induce multiple ovulations (superovulation) have evolved from superstimulatory protocols that depended upon detection of oestrus to treatments that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-timed AI (FTAI). The protocols associated with FTAI facilitate animal handling and produce at least as many viably embryos as conventional treatment protocols that required detection of oestrus. Recent knowledge regarding LH receptors (LHR) and follicular development can be applied to improve embryo transfer protocols. In fact, improvements in the superstimulatory treatment called the ‘P-36 protocol', which include hormones that stimulate LHR, indicate that adjustments related to LHR availability may increase bovine embryo yield compared with conventional protocols based on the detection of oestrus. [ABSTRACT FROM AUTHOR]

Media designed for the recovery, holding and cryopreservation of bovine and equine embryos are available from several commercial sources. In years past, some of these media contained bovine serum, although inclusion of serum in embryo transfer media is now largely discontinued due to issues relating to storage and biosecurity. Currently, bovine serum albumin (BSA) is included in most commercially manufactured media intended for use in embryo transfer (ET). Although BSA poses less risk than serum for the transmission of infectious disease, its inclusion still entails risk of viral contamination. The present review briefly describes the various components of ET media and the development of efficacious ET media containing no products of animal origin. An evaluation of the efficacy of recovery, holding, slow controlled freezing and vitrification media in both research and commercial ET embryo transfer settings is also presented. [ABSTRACT FROM AUTHOR]

There is continuing need to simplify bovine superovulation protocols without compromising embryo production. The control of follicular wave emergence and ovulation has facilitated donor management, but the most commonly used treatment, oestradiol, cannot be used in many parts of the world and mechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include gonadotrophin-releasing hormone (GnRH) or LH, but efficacy in groups of randomly cycling animals is variable. Another alternative is to increase the response to GnRH by inducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferable embryos following superovulation during the first follicular wave did not differ from that achieved 4 days after oestradiol benzoate and progesterone. To further simplify superovulation, FSH has been administered as a single intramuscular injection. Superovulation of beef donors with a single intramuscular injection of Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) diluted in a slow-release formulation resulted in embryo production comparable to that obtained using the traditional twice-daily protocol. The single intramuscular injection has the potential to reduce labour and handling and may be useful when handling stress is an impediment to success. These alternatives provide ways of facilitating widespread application of embryo transfer technologies. [ABSTRACT FROM AUTHOR]

Development of the post-hatching conceptus in ruminants involves a period of morphological expansion that is driven by complex interactions between the conceptus and its intrauterine environment. As a result of these interactions, endometrial physiology is altered, leading to establishment of the pregnancy and continued development of the placenta. Disruption of normal fetal and placental development can occur when embryos are exposed to manipulations in vitroor when inappropriate endocrine sequencing occurs in vivoduring the pre- and peri-implantation periods. The present review addresses the development of the post-hatching bovine conceptus, its interactions with the maternal system and changes in development that can occur as a result of in vivoand in vitromanipulations of the bovine embryo. [ABSTRACT FROM AUTHOR]

For more than 40 years, practitioners have sought to improve all aspects of commercial bovine embryo transfer. The development of new technologies for this industry has been substantial, with recent focus on cryopreservation techniques and the in vitroproduction of embryos fertilised with sexed spermatozoa. When these and other new technologies are developed, the following questions remain: (1) is said technology regulated or does it require licensing; and (2) is it applicable and, if so, is it financially feasible? Computer access to published research and the advancement of data software programs conducive to the industry for data procurement have been essential for helping practitioners answer these questions by enhancing their ability to analyse and apply data. The focus of the present paper is to aid commercial embryo transfer practitioners in determining new technologies that are available and whether they can be implemented effectively, benefiting their programs. [ABSTRACT FROM AUTHOR]

Data on fertilisation and embryo quality in dairy cattle are presented and the main factors responsible for the low fertility of single-ovulating lactating cows and embryo yield in superovulated dairy cattle are highlighted. During the past 50 years, the fertility in high-producing lactating dairy cattle has decreased as milk production increased. Recent data show conception rates to first service to be approximately 32% in lactating cows, whereas in heifers it has remained above 50%. Fertilisation does not seem to be the principal factor responsible for the low fertility in single-ovulating cows, because it has remained above 80%. Conversely, early embryonic development is impaired in high-producing dairy cows, as observed by most embryonic losses occurring during the first week after fertilisation. However, in superovulated dairy cattle, although fertilisation failure is more pronounced, averaging approximately 45%, the percentage of fertilised embryos viable at 1 week is quite high (>70%). Among the multifactorial causes of low fertility in lactating dairy cows, high feed intake associated with low concentrations of circulating steroids may contribute substantially to reduced embryo quality. Fertilisation failure in superovulated cattle may be a consequence of inappropriate gamete transport due to hormonal imbalances. [ABSTRACT FROM AUTHOR]

Numerous studies have shown that it is possible to manipulate follicular and luteal dynamics, thereby eliminating the need for oestrus detection in embryo transfer (ET) programmes. Fixed-time ET (FTET) protocols are based on the use of gonadotrophin-releasing hormone (GnRH) and prostaglandin (PG) F or progesterone/progestogen (P4)-releasing devices and oestradiol. The FTET protocols increases the proportion of recipients transferred, and therefore pregnancy rates, compared with the use of PGF followed by ET 7 days after oestrus. Furthermore, the addition of equine chorionic gonadotrophin (eCG) to the P4 and oestradiol-based FTET protocols results in an even higher proportion of recipients transferred, and thus higher pregnancy rates. The beneficial effect of eCG treatment may be related to increased growth of the dominant follicle and increased plasma P4 concentrations during the subsequent luteal phase. In Bos taurus× Bos indicusrecipients, pregnancy rates were positively correlated with the diameter of the corpus luteum (CL) and the number of CL at ET. When repeat-breeder Holstein cows were used as recipients, FTET protocols increased number of recipients transferred and pregnancy rates compared with the traditional PGF-based synchronisation protocols. In conclusion, the use of FTET protocols eliminates the need for the detection of oestrus and results in a greater proportion of recipients transferred and satisfactory pregnancy rates. Thus, FTET optimises the use of recipients, reducing labour and animal handling and facilitating the use of ET. [ABSTRACT FROM AUTHOR]

One limitation to the use of in vitro-produced embryos in cattle production systems is the fact that pregnancy rates after transfer to recipients are typically lower than when embryos produced in vivoare transferred. Conceptually, the oocyte and spermatozoon from which the embryo is derived could affect competence for post-transfer survival. There are sire differences in embryonic survival after transfer, but there is little evidence that an embryo's ability to establish pregnancy is determined by sex sorting of spermatozoa by flow cytometry. The role of the source of the oocyte as a determinant of embryonic survival after transfer has not been examined carefully. Conditions for embryo culture after fertilisation can have an impact on the ability of the embryo to establish pregnancy following transfer. Among the specific molecules produced in the reproductive tract of the cow that have been shown to improve competence of in vitro-produced embryos for post-transfer survival are colony-stimulating factor 2, insulin-like growth factor-1 (for recipients exposed to heat stress) and hyaluronan (for less-advanced embryos). There is also a report that embryo competence for post-transfer survival can be improved by inclusion of a carbon-activated air filtration system in the incubator used to culture embryos. Progress in developing culture systems to improve embryonic competence for survival after transfer would be hastened by the development of in vitroassays that accurately predict the potential of an embryo to establish pregnancy after transfer. A group of 52 genes has been identified that are differentially expressed in embryos that developed to term v.embryos that did not establish pregnancy. Perhaps a gene microarray consisting of these genes, alone or in combination with other genes, could be used to screen embryos for competence to establish pregnancy. [ABSTRACT FROM AUTHOR]

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2–8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis. [ABSTRACT FROM AUTHOR]

Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitromatured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 μmLG100268 (LG; a retinoid X receptor agonist), 0.7 μmall-transretinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis. [ABSTRACT FROM AUTHOR]

Gamete recognition and binding of spermatozoa to the oviduct are carbohydrate-mediated processes in which several glycosidases are thought to have a role, although this has not been demonstrated unequivocally. Oviducal fluid is the biological milieu in which fertilisation and early embryo development take place, but the enzyme composition of oviducal fluid is largely unknown. The aim of the present study was to determine glycosidase activity and protein content in bovine oviducal fluid (bOF) and the volume of fluid collected per oviduct. Oviducts obtained from a slaughterhouse were classified as either in the follicular or luteal phase on the basis of ovarian luteal morphology. Oviducal fluid was aspirated, centrifuged and the volume determined. Samples were then frozen until assay. Substrates conjugated to 4-methylumbelliferyl were used to screen for the activity of seven glycosidases at pH 7.2. The results indicate that bOF has α-l-fucosidase, β-N-acetyl-glucosaminidase, β-d-galactosidase, α-d-mannosidase and β-N-acetyl-galactosaminidase activity during both phases of the cycle, with the specific activity of the latter two enzymes being higher during the follicular phase. There was no N-acetyl-neuraminidase or α-d-galactosidase activity detected in bOF at either phase of the oestrous cycle at pH 7.2, but activity for both glycosidases was detected at pH 4.4. There were no differences in protein concentration or the volume of bOF collected between the two phases of the cycle. These findings indicate that oviducal fluid exhibits glycosidase activity, with specific variations throughout the oestrous cycle, suggesting that these enzymes play a role in carbohydrate-mediated events. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of a novel density gradient centrifugation (DGC) treatment using recombinant trypsin on the in vivofertilising capability of bovine spermatozoa compared with a standard method. In Trial 1, semen collected from Boran and Ankole (Bos indicus) bulls was treated either with a silane-coated silica particle colloid formulated for humans with a recombinant trypsin or processed using a standard method (dilution in an egg yolk-based diluent). Semen processed by the two methods was used to artificially inseminate (AI) superovulated cattle. Day 7 embryos were flushed and assessed for fertilisation rates and embryo quality. Trial 2 used a trypsinised silane-coated silica particle colloid formulated specifically for bovine semen. Trial 1 resulted in significantly higher fertilisation rates using the trypsinised human DGC treatment than cows inseminated using the standard method (75.2% v.67%, respectively; P 0.05). Results for Trial 2 indicated that cows inseminated with the trypsinised bovine DGC treatment had significantly increased fertilisation rates compared with the standard method (88.4% v.63.1%, respectively; P< 0.01) and had significantly higher numbers of transferable-quality embryos (70.3% v.51.8%, respectively; P< 0.01). In summary, bovine sperm treatment before AI by DGC and recombinant trypsin increases fertilisation rates and can result in more transferable-quality embryos compared with standard methods. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the effect of maturation for 16 v. 24 h on the kinetics of development and the sex ratio of bovine embryos. Oocytes were inseminated at 16 or 24 h after the beginning of maturation using frozen–thawed bull semen. Two-cell embryos at 24, 28, 32, 36, 40, 44 and 48 h post-insemination (hpi) and blastocysts at Days 6, 7 and 8 from both groups were snap-frozen individually and stored at –80°C until determination of embryo sex. Insemination at 16 h resulted in a lower cleavage rate at 48 hpi than insemination at 24 h (70.6% v. 77.1%, respectively, P < 0.05). In terms of the evolution of cleavage divisions, insemination at 24 h resulted in a typical pattern of cleavage such that by 32 hpi, ~58% of presumptive zygotes had cleaved. In contrast, first cleavage following insemination at 16 h was significantly slower such that by 32 hpi, ~35% of presumptive zygotes had cleaved. Duration of IVM did not affect blastocyst yield (~37%). The overall sex ratio of 2-cell embryos at 48 hpi differed from 1 : 1 in favour of males in both groups (24 h: 55.9 v. 44.1%; 16 h: 59.1 v. 40.9%, P < 0.05). Similarly, the overall sex ratio of blastocysts differed from 1 : 1 in both groups (24 h: 59.7 v. 40.3%; 16 h: 58.5 v. 41.5%, P < 0.05). In conclusion, timing of gamete interaction and maturity of the oocyte at the time of the interaction can affect the kinetics of the early cleavage divisions but has no effect on the sex ratio of the embryos produce. [ABSTRACT FROM AUTHOR]

The effect of s-nitroso-n-acetyl-l,l-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10?3m SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P 0.05), but superior to the other treatment groups (P 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10?7m SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development. [ABSTRACT FROM AUTHOR]

Higher systemic progesterone in the immediate post-conception period is associated with an increase in embryonic growth rate, interferon-tau production and pregnancy rate in cattle. The objective of this study was to examine the effect of increasing progesterone concentration on Day 3 on subsequent embryo survival and development. Oestrus (Day 0) was synchronised in beef-cross heifers (n = 210) and approximately two-thirds of the heifers were inseminated with semen from a proven sire, while the remainder were not inseminated. In order to produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the oestrous cycle, which was left in situ until slaughter. The four treatment groups were: (i) pregnant, high progesterone; (ii) pregnant, normal progesterone; (iii) non-pregnant, high progesterone; and (iv) non-pregnant, normal progesterone. Animals were blood-sampled twice daily from Days 0 to 8 and once daily thereafter until slaughter on Days 5, 7, 13 or 16, corresponding to the 16-cell stage, the blastocyst stage, the beginning of elongation and the day of maternal recognition of pregnancy, respectively. Embryos were recovered by flushing the tract with phosphate-buffered saline and characterised by stage of development and, in the case of Days 13 and 16, measured. Data were analysed by mixed models ANOVA, Chi-square analysis and Student''s t-test where appropriate. Insertion of a PRID on Day 3 increased (P < 0.05) progesterone concentrations from Day 3.5 onwards. There was no difference between treatments in the proportion of embryos at the expected stage of development on Days 5 or 7 (P > 0.05). While not significantly different, the proportion of viable embryos recovered was numerically greater in the high progesterone group on both Day 13 (58 v. 43%) and Day 16 (90 v. 50%). Elevation of progesterone significantly increased embryonic length on Day 13 (2.24 0.51 mm v. 1.15 0.16 mm, P = 0.034) and Day 16 (14.06 1.18 cm v. 5.97 1.18 cm, P = 0.012). In conclusion, insertion of a PRID on Day 3 of the oestrous cycle increased serum progesterone concentrations on subsequent days, which, while having no phenotypic effect on embryonic development on Days 5 or 7, was associated with an increase in embryonic size on Days 13 and 16. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 1.28 and 10.30 0.51 ng mL?1, respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 0.45 mm2 (n = 28) compared with 1.66 0.38 mm2 (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos. [ABSTRACT FROM AUTHOR]

The main objective of this study was to compare pregnancy and calving rates following transfer of in-vitro-produced fresh river and F1 (river swamp) buffalo embryos in recipients synchronised by Ovsynch protocol or following natural oestrus. River embryos were produced from cumulus?oocyte complexes (COCs) derived by ovum pick up (OPU) on 40 Murrah and Nili-Ravi donor buffaloes over a twice-weekly collection schedule for 120 single OPUs. F1 embryos were produced by fertilisation of swamp COCs recovered from abattoir ovaries coincubated with river sperm cells. Both groups of embryos were produced following the same protocol for in vitro production. With regard to the OPU source of COCs, 923 antral follicles were punctured and 647 COCs were recovered (70%). From 462 selected COCs for IVM, 257 (55.6%) cleaved zygotes were recorded leading to 93 blastocysts (20.1%). In total, 590 swamp COCs were aspirated from abattoir ovaries and 476 were selected for IVM leading to 270 (56.7%) cleaved zygotes and resulting in 137 blastocysts (28.8%). River and F1 embryos were transferred between Day 6 to 7 of in vitro development, corresponding to blastocyst?expanding blastocyst, into F1 recipients synchronised by Ovsynch and swamp buffaloes following natural oestrus, respectively, each of them receiving two embryos. According to palpation per rectum of the ovaries at the time of embryo transfer, 26 of the 47 (55.3%) F1 recipients synchronised by Ovsynch were considered suitable for transfer, resulting in seven pregnancies (26.9%) and four calvings (15.3%) owing to three abortions occurring between 2 and 3 months of pregnancy. In total, 29 swamp recipients following natural oestrus were judged suitable as recipients, resulting in 12 pregnancies (41.4%) and 10 calvings (34.5%) owing to two abortions at 2 and 3 months of gestation respectively. Pregnancy and calving rates following transfer of river and F1 embryos were similar. Likewise, weight at birth of calves derived from transfer of river and F1 embryos was not different: 30.5 1.4 and 32.9 2.4 respectively. Pregnancy and calving rates following AI in a group of river and swamp buffaloes considered for reference in this study were similar to recipients carrying in-vitro-produced embryos. Collectively, no apparent postnatal complications were recorded in resulting live calves. [ABSTRACT FROM AUTHOR]

An abstract of the study "Influence of Breed Dominance on Maturation Competence, Fertilization, and Development In Vitro of Bovine Oocytes," by J. Pelaez and colleagues is presented.

An abstract of the study "Zona Pellucida Tagging With Barcodes Allows the Traceability of Bovine Embryos Cultured in Group," by S. Novo and colleagues is presented.

An abstract of the study "Resveratrol During In Vitro Culture Improves Cryotolerance of In Vitro Produced Bovine Embryos," by G. Zullo and colleagues is presented.

An abstract of the study "Fatty Acid β-Oxidation Regulates Bovine Embryo Oxygen Metabolism," by P. J. McKeegan and colleagues is presented.

An abstract of the study "Effect of a Novel Bovine Embryo Culture Medium to Improve Blastocyst Development," by M. A. Achilles and colleagues is presented.

An abstract of the study "Cysteine and Catalase Supplementation for 7 Days During In Vitro Culture Impairs Bovine Embryo Development," by N. A. S. Rocha and colleagues is presented.

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL-1 of pyruvate, 75 μg mL-1 of gentamicin, 20 μg mL-1 of FSH and 10 IU mL-1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL-1 of fatty acid-free BSA, 2 μL mL-1 of pyruvate, 75 μg mL-1 of gentamicin, 11 μg mL-1 of heparin and 44 μL mL-1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP. [ABSTRACT FROM AUTHOR]

More than 300 000 embryos have been transferred all over the world (Stroud 2010 IETS Newsl. 27(4), 11-21). We have reported that embryos that showed the abnormal cleavage pattern at the first cell division can develop to the blastocyst stage (Somfai et al. 2010 J. Reprod. Dev. 56, 200-207). However, we have limited knowledge about the consequences of the pattern of first embryonic cleavage on their post-transfer developmental competence. The present study was conducted to determine the developmental competence of bovine blastocysts showing different cleavage patterns at their first cell division. Cumulus-oocyte complexes were collected by ovum pickup from Japanese Black cows and were subjected to in vitro maturation and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52, S19-S29 suppl). Inseminated oocytes were cultured in CR1aa medium supplemented with 5% calf serum covered by mineral oil at 38.5°C in 5% CO2 in air with micro-droplets or 5% CO2, 5% O2 and 90% N2. The kinetics of embryo development were analysed by time-lapse cinematography for 168 h after IVF by using a Cultured Cell Monitoring System (CCM-M1.4ZS, Astec, Fukuoka, Japan). A total of 673 photographs of each embryo were taken (1 photograph in every 15 min) during in vitro culture. Image stacks were analysed by the CCM-M1.4 software. Embryos were classified in 5 groups according to the pattern of first cleavage as normal cleavage (NC), direct cleavage from 1 cell to 3 to 4 blastomeres (3-4BL), unequal blastomeres (UB), multiple fragments (MF) and protrusion formation (PT). Blastocysts developing from each group were transferred into the ipsilateral uterine horn of each synchronized recipient on Day 7 or 8 after oestrus. Data on conception at Day 60, abortion and delivery were then recorded. Data were analysed by chi-square test and Student's t-test. In total, 43 embryos were transferred, 17 conceptions (39.5%) were established and 16 recipients (94.1%) were delivered. Only 1 abortion was detected at Day 223 in the NC group. The highest conception rate was observed in the NC group (55%, n = 20) and the 3-4BL (n = 12), UB (n = 6) and PT (n = 3) groups showed similar conception rates of 33.3% (1 implanted embryo belonged to 2 classes in UB and PT) and none of the embryos derived from the MF group (n = 3) could cause conception. There was a significant difference (P < 0.05) in conception rates between the NC group and totals of each of the other cleavage groups. No significant difference was found in gestation lengths and birth weights between the NC group (282.2 ± 4.4 days, 30.6 ± 3.8 kg, respectively) and totals of each of the other cleavage groups (282.8 ± 5.3 days, 30.3 ± 1.9 kg, respectively). These results indicate that embryos showing abnormal cleavage patterns at first cell division can develop to normal calves with normal gestation lengths and birth weights; however, their post-transfer viability is lower than for NC embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives. [ABSTRACT FROM AUTHOR]

In vitro-produced Bos taurus indicus (zebu) and Bos taurus indicus × Bos taurus taurus (cross-bred) embryos behave differently when vitrified. The present experiment aimed to examine the effect of vitrification on embryos produced in the presence or absence of FCS. Cumulus-oocyte complexes (COC) were matured in TCM-199 and fertilized in human tubal fluid medium with frozen Nelore bull semen. On Day 1 (Day 0 = IVF), presumptive zygotes were cultured with SOFaa + BSA in the presence of FCS (Group 2.5%) or in the absence of FCS (Group 0%) until Day 7. The cleavage was analysed on Day 3 and the blastocyst rate on Day 7. Blastocysts were vitrified and, after warming (Campos-Chillòn et al. 2006) the viability was evaluated. Data were analysed with ANOVA, using the general linear model (GLM) of SAS (SAS Inst Inc., Cary, NC, USA). Sources of variation in the model included FCS concentration and first-order interactions; all factors were considered fixed effects. The arcsine transformation (√y/100) was applied to percentage data. If the ANOVA was significant, means were separated using the Tukey test. There was no difference in cleavage (for zebu embryos: Group 0%: 87.2 ± 6.8; Group 2.5%: 87.4 ± 9.5; for cross-bred embryos: Group 0%: 79.6 ± 11.9; Group 2.5%: 73.1 ± 13.7; P > 0.05). On the other hand, zebu embryos cultured in the presence of FCS reached blastocysts at a higher rate than cross-bred embryos in the absence of FCS (for zebu embryos: Group 0%: 33.3 ± 12.4ab; Group 2.5%: 46.8 ± 13.2; for cross-bred embryos: Group 0%: 21.8 ± 8.3; Group 2.5%: 33.6 ± 10.1ab; P < 0.05). After vitrification and warming, no significant differences in re-expansion rate (zebu embryos: Group 0%: 82.7 ± 13.1; Group 2.5%: 75.0 ± 9.8; cross-bred embryos: Group 0%: 93.7 ± 8.8; Group 2.5%: 84.1 ± 11.3; P > 0.05) and cell number per embryo (zebu embryos: Group 0%: 65.1 ± 34.7; Group 2.5%: 42.6 ± 17.2; cross-bred embryos: Group 0%: 64.3 ± 44.2; Group 2.5%: 52.0 ± 31.5; P > 0.05) between species groups and within species were seen. However for zebu embryos, Group 0% showed a lower damaged cell rate than Group 2.5%. The same effect was not observed in the cross-bred embryos (zebu embryos: Group 0%: 20.3 ± 22.7; Group 2.5%: 63.3 ± 27.0; cross-bred embryos: Group 0%: 25.4 ± 24.3cd; Group 2.5%: 45.8 ± 34.6cd; P < 0.05). The addition of 2.5% FCS had a higher deleterious effect on zebu embryos than cross-bred (zebu × taurine) embryos after vitrification. These results also reinforce the species differences observed between zebu and cross-bred, as they behaved differently in relation to the addition of FCS in the culture medium and in relation to their cryopreservation sensitivity. Supported by FAPESP 10/50410-2. [ABSTRACT FROM AUTHOR]

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitromatured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50ngmL-1of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitrodevelopment of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P<0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope. [ABSTRACT FROM AUTHOR]

Bos taurus indicusfemales provide a greater in vivooocyte recovery (2-4 times more) in comparision with Bos taurus taurus. This aspect has strongly contributed for the success of the embryo industry in Brazil. Little information is available to explain this difference. Our objective was to test the hypothesis that the difference in oocyte yield is a result of higher numbers of preantral follicles in the ovaries of B. indicusfemales. Ovaries (n= 44) of fetuses aging from 180 to 240 days and of heifers from 20 and 24 months were collected from Bos taurus indicus(Nelore) and Bos taurus taurus(Aberdeen Angus) females at a slaughterhouse. Fetal age was estimated from the crown-rump length (Evans HE and Sack WO 1973 Anat. Hist. Embryol. 2, 11-45). Ovaries from Nelore fetuses (n= 10) and heifers (n= 12) and Aberdeen Angus fetuses (n= 10) and heifers (n= 12) were cut longitudinally into two halves, fixed in Bouin''s solution, and processed for histological evaluation. The ovarian halves were dehydrated in alcohol, cleared with xylene, embbebed in paraffin, and serially sectioned at 7 μm. Every 120th section was mounted and stained with periodic acid Schiff and hematoxylin. The number of preantral follicles was estimated through the counting of follicles in each section using the nucleus of the oocyte as a marker and a correction factor (Gougeon A and Chainy GBN 1987 J. Reprod. Fertil. 81, 433-442). Only 1 ovary per female was analyzed. Preantral follicles were classified according to the developmental stage as primordial (1 layer of flattened granulosa cells surrounding the oocyte), primary (1 layer of cuboidal granulosa cells), or secondary (2 or more layers of cuboidal granulosa cells), and as normal or degenerated according to their morphological appearance. The number of preantral follicles was not normally distributed and was compared using the Mann-Whitney test. There was no difference (P> 0.05) between the average number of preantral follicles in the ovaries of Bos indicusand Bos taurusfemales. The average number of preantral follicles per female was 143 929 ± 253 (mean ± SD) and 285 155 ± 570 for Bos taurusand Bos indicusfetuses, respectively, and 76 851 ± 280 and 109 673 ± 293 for Bos taurus andBos indicus heifers (P> 0.05), respectively. A large variation in numbers of preantral follicles was observed among individuals within the same category and between breeds. Our results suggest that there might be differences in mechanisms controlling follicle development after the preantral stage accounting for the greater oocyte yield from Bos indicusfemales. This work was supported by grants from the Universidad Nacional del Litoral (CAI+D program), Universidad Nacional de Lomas de Zamora, the Argentine National Agency for the Promotion of Science and Technology (ANPCyT), and the Argentine National Council for Science and Technology (CONICET). [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids seem to exert an extra-caloric positive effect on ruminant reproduction, although the reasons for that are still unclear. Although some studies have detected a positive effect of feeding unsaturated fatty acids on embryo development of superovulated Bos tauruscattle (Thangavelu et al.2007 Theriogenology 68, 949-957), others have not (Petit et al.2008 J. Dairy Sci. 91, 1786-1790). Our hypothesis was that, although number and quality of embryos from superovulated heifers would not be affected by diet, supplemental fat would improve embryo cryotolerance. Therefore, this study evaluated superovulatory response and embryo production, as well as cryotolerance of embryos cryopreserved through freezing or vitrification in Nellore heifers supplemented with rumen-protected fat. Forty heifers (24 to 36 mo old) were kept in pasture and randomly divided into 2 experimental groups according to supplemental source [F = concentrate with rumen-protected fat (100 g/d of Megalac-E®) and C = control, without fat supplementation]. Supplements were formulated to be isocaloric and isoproteic. Each female underwent both treatments in a cross-over design with approximately 68 d between replicates. After 50 d of feeding, emergence of the wave was synchronized with the aid of hormones to initiate the superovulation protocol. Recovered embryos were frozen or vitrified, and subsequently in vitroembryo development evaluation was accomplished. Data were analyzed using generalized linear models. There was no difference between F and C groups (P> 0.10) regarding superstimulatory response, number of total embryos/ova, viable embryos, degenerate embryos, or unfertilized oocytes recovered. However, group C had a greater superovulatory response than F (18.0 ± 1.3 v.15.7 ± 1.2 CL; P= 0.06). Group C embryos presented greater hatching rate, independently of the cryopreservation method, at 48 h (33.1 ± 4.0%; n= 148 v.17.3 ± 3.3%; n= 137; P= 0.009) and at 72 h (44.3 ± 4.2%; n= 148 v.30.9 ± 4.0%; n= 137; P= 0.04) of in vitroculture. Under the conditions of the present study, supplementation with protected fat did not affect superstimulatory response and quantity or quality of embryos. However embryos from the F group were less tolerant to cryopreservation. Financial support from FAPESP, EMBRAPA, Arm & Hammer, Integral Produbon, and Pfizer of Brazil. [ABSTRACT FROM AUTHOR]

In this study we evaluated mitochondrial distribution of individual bovine embryos after IVF, parthenogenetic activation (PG), and somatic cell nuclear transfer (SCNT). COCs obtained from slaughterhouse ovaries were matured in vitrofor at least 18 h in TCM-199 supplemented with hormones, and then divided into 3 groups. SCNT and PG oocytes were stripped by vortexing in HEPES-HECM (hamster embryo culture medium) medium (HH) containing hyaluronidase and metaphase II (MII) oocytes selected by visualization of a polar body. The PG group oocytes were exposed 24h post-maturation to 5 μMionomycin in HH for 4 min, then rinsed 3 times in HH and allocated to a 4-h culture in 2 mMDMAP in KSOM for activation. The SCNT group oocytes were included in a nuclear transfer procedure performed as previously described (Ross et al.2006 Biotechniques 41, 741-750). Activation was performed as described for the PG group. The IVF group COCs were co-incubated for 20 h with 106spermatozoa/mL in IVF-TALP supplemented with heparin. To label mitochondria, 1 mMMitoTracker CMXRos Red (Molecular Probes, Eugene, OR, USA) was added to HH at a final concentration of 0.3 μM. Samples were cultured for 15 min, washed in HH, placed in a glass-bottomed 35-mm Petri dish, and then observed and live photographed by using a spinning disk confocal microscope (Nikon Eclipse TE2000-E + CARV Confocal) equipped with a Cascade 512 B camera (Roper Scientific, Tucson, AZ, USA) using a Nikon 40×, 1.3 NA oil objective lens. Z series images were taken acquiring 15 focal planes at 10-μm intervals. Analysis was performed using Metamorph software. Samples were taken at pronuclear, 4 cell, and morula stages. Each sample was classified for its mitochondrial localization in pericytoplasm, cytoplasm, and perinuclear. Data was analyzed by proc glimmix (SAS, Cary, NC, USA). Significance was set at P< 0.05. A similar pericytoplasmic distribution of mitochondria for all treatments up to the 4-cell stage was observed. At the pronucelar stage, mitochondria distribution was mostly pericytoplasmic, changing to cytoplasmic at the 4-cell stage. At the morula stage there was a significantly higher number of embryos with perinuclear distribution in IVF than in PG and SCNT embryos (Table 1). Our findings demonstrate that mitochondrial reorganization differs in fertilized more-developed embryos compared with their activated counterparts. This may have implications for further embryo development, mainly after SCNT. Table 1.Mitochondria distribution in fertilized, parthenogenetic, and cloned bovine embryos [ABSTRACT FROM AUTHOR]

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitrodevelopment of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al.2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitrodevelopment, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivodevelopment and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitroand in vivodevelopment of cloned bovine embryosThis study was supported by FAPESP and CAPES, Brazil. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNA) are key regulators of gene expression in vertebrate development; however, little information exists about their expression in pre-implantation mammalian embryos. We studied the expression of miRNA in bovine cloned and IVF-elongated embryos using microarrays and quantitative RT-PCR (qRT-PCR). Day-7 IVF or handmade cloned blastocysts from an adult cell line that previously yielded live cloned calves were transferred to synchronized heifers and recovered at Day 17 as elongated embryos. MicroRNAs were purified using miRVANA kit+Flash Page fractionator column (Ambion, Austin, TX, USA) coupled to amine reactive dyes CyDye™ or used in qRT-PCR. Arrays were custom-made from 271 mice, human, and rat and 104 predicted miRNAs. After overnight hybridization, slides were scanned and data analyzed. Only miRNAs showing hybridization in 3 out of 6 features per slide were considered to be expressed. MicroRNAs were simultaneously converted to complementary (c)DNA and elongated using miRNA-specific DNA-oligo with 5′ overhang and primer extension by reverse transcription, followed by SYBR Green qPCR with another miRNA-specific primer and an excess of universal forward and reverse primers. The qPCR were normalized against 5S rRNA. Data were analyzed using Kruskal-Wallis nonparametric test for P-values < 0.05. Cloned (n= 48) and IVF (n= 28) blastocysts were transferred into 10 and 4 surrogate cattle, and 42 elongated embryos were recovered; 13 of these with embryonic disc were used for miRNA extraction and analysis. In experiment 1, miRNA profiling (1 slide) from 4 cloned embryos and donor cells yielded 34 and 22 miRNAs expressed in the embryos and cells, respectively. In experiment 2 (differential expression; 3 replicates), 39 miRNAs were expressed in cloned embryos and 32 in IVF embryos. Fifteen of these miRNAs were differentially expressed: 7 were up-regulated in the cloned elongated embryos and 8 in the elongated IVF embryos. The profiles of miRNA expression of cloned embryos, IVF embryos, and the donor cells were compared and reprogramming patterns analyzed. Reprogramming means similar expression among cloned and IVF embryos, but different from the somatic cells. Overall, 50 miRNAs were subjected to nuclear reprogramming; 31 (62%) were reprogrammed correctly, 10 aberrantly (20%), and 9 were not reprogrammed (18%). Most of the aberrantly reprogrammed miRNAs (90%) were switched on after nucleus transfer. Some of the reprogrammed miRNAs clustered in the same genomic location. Based on microarray data, 3 miRNAs over-expressed in the IVF embryos (let7-b, miR-200c, and miR-24), and 1 equally expressed (miR-16) and 2 over-expressed in the cloned embryos (miR-21 and miR-103) were analyzed by qRT-PCR. Expression of all the assayed miRNAs except miR-200c correlated adequately with microarray data (Pearson coefficient of correlation R2= 0.9; P= 0.01). Here we present the first description of miRNA expression and reprogramming in bovine elongated cloned embryos. This can have profound implications for the understanding of nuclear reprogramming in somatic cloning. [ABSTRACT FROM AUTHOR]

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P> 0.05) between control and recloned groups regarding fusion rate (n= 550, 62.22% and n= 189, 57.25%; respectively) or cleavage rate (n= 383, 69.45% and n= 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P< 0.01) compared with the control group (n= 51, 26.98%, and n= 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n= 6, 15.38% and n= 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil. [ABSTRACT FROM AUTHOR]