Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America,and personal reminisces

After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses.     

Development of interspecific Solanum lycopersicum and screening for Tospovirus resistance

Tospovirus has emerged as a serious viral pathogen for several crops including tomato. The tomato production is being severely affected worldwide by Tospovirus. Some reports have been published about the association of plant virus and development of human disease either by direct or indirect consumption. Resistance to this virus has been identified as good source in wild tomato species (Lycopersicum peruvianum). But the introgression of resistance genes into cultivated tomato lines and the development of interspecific hybrid are hampered due to incompatibility, fertilization barriers and embryo abortion. But this barrier has been broken by applying the embryo rescue methods. This study describes the development of interspecific hybrid tomato plants by highly efficient embryo rescue method and screening for Tospovirus resistance. The interspecific hybrid tomato plants were developed by making a cross between wild tomato species (L. peruvianum) and cultivated tomato (Solanum lycopersicum). The immature embryos were cultured in standardized medium and interspecific hybrids were developed from embryogenic callus. The immature embryos excised from 7 to 35 days old fruits were used for embryo rescue and 31 days old embryos showed very good germination capabilities and produced the highest number of plants. Developed plants were hardened enough and shifted to green house. The hybrid nature of interspecific plants was further confirmed by comparing the morphological characters from their parents. The F1, F2 and F3 plants were found to have varying characters especially for leaf type, color of stem, fruits, size, shapes and they were further screened for virus resistance both in lab and open field followed by Enzyme linked Immunosorbant Assay confirmation. Finally, a total of 11 resistant plants were selected bearing red color fruits with desired shape and size.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.     

Improving post-transfer survival of bovine embryos produced in vitro: actions of insulin-like growth factor-1, colony stimulating factor-2 and hyaluronan

Technologies for in vitro embryo production have the potential to enhance the efficiency of cattle production systems. However, utilization of in vitro-produced embryos for transfer remains limited throughout much of the world. Despite improvements over the past two decades, problems associated with the production of bovine embryos in vitro still exist which limit the widespread commercial application of this technology. In particular, bovine embryos produced in vitro have a reduced capacity to establish and maintain pregnancy as compared with their in vivo-derived counterparts. Embryo competence for survival following transfer is improved by in vivo culture in the sheep oviduct, thus indicating that standard embryo culture conditions are sub-optimal. Therefore, one strategy to improve post-transfer survival is to modify embryo culture media to more closely mimic the in vivo microenvironment. The maternal environment in which the bovine embryo develops in vivo contains various growth factors, cytokines, hormones, and other regulatory molecules. In addition to affecting bovine embryo development in vitro, recent research indicates that embryo competence for survival following transfer can also be improved when such molecules are added to embryo culture medium. Among the specific molecules that can increase post-transfer embryo survival are insulin-like growth factor-1 (IGF-1), colony stimulating factor-2 (CSF-2) and hyaluronan. This paper will review the effects IGF-1, CSF-2 and hyaluronan on post-culture embryo viability and discuss the potential mechanisms through which each of these molecules improves post-transfer survival.     

Developmental consequences of association with a photosynthetic substrate for encapsulated embryos of an intertidal gastropod

Aggregation of embryos in clutches that lack internal circulation can increase the risk of hypoxia by limiting gas exchange. As a result, limits on oxygen solubility and diffusion in water can constrain the size and embryo concentration of aquatic egg clutches. Hypoxia in egg masses can slow embryo development, increase mortality, and reduce size at hatching. The risk of hypoxia for embryos, however, can be reduced by association with photosynthetic organisms. We examined whether embryo development in egg ribbons of the cephalaspidean mollusk Haminoea vesicula is significantly influenced by oviposition on eelgrass (Zostera marina). Association with the photosynthetic substrate had marked effects on development relative to association with non-photosynthetic substrates, and the direction of these effects was mediated by light conditions. Under intermediate and high light levels, association with eelgrass accelerated embryo development, while under dim light, the presence of the macrophyte increased development rate and reduced hatchling shell size. Benefits of association with eelgrass at higher light levels likely result from oxygen production by eelgrass photosynthesis, while we attribute costs under low light to oxygen depletion by eelgrass respiration. Association with Z. marina also limited microphyte growth in egg ribbons of H. vesicula. In the field, measurements of light attenuation within an eelgrass bed showed that conditions under which benefits accrue to embryos are ecologically relevant and correspond to spatial patterns of oviposition on eelgrass in the field. The choice of a photosynthetic oviposition substrate under appropriate light conditions can improve embryo fitness by accelerating embryo development without compromising hatchling size and by reducing the potential for excessive and harmful fouling by microphytes.     

Possible effects of pituitary adenylate cyclase activating polypeptide (PACAP) on early embryo implantation marker HB-EGF in mouse

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated as a hypothalamic neuropeptide stimulating adenylate cyclase activity. Besides its neuroprotective effects, numerous data proved its role in reproductive processes. However, there are limited data on its role in preimplantation embryo development and implantation. Our aim was to analyse the mRNA expression of Adcyap1 (coding region of PACAP) and Hbegf [coding region of HB-EGF (Heparin-binding EGF-like growth factor)] in embryos and pregnant uterus to investigate the possible correlation between them. Eight-week-old BDF1 mice were superovulated and subsequently mated overnight or left in their cage after hCG treatment. Day4 embryos were flushed from mated females. After morphological analysis, Adcyap1 and Hbegf gene expression of embryos and uterine tissues was assessed with qPCR.Our results showed significantly higher Adcyap1 and Hbegf mRNA levels in females producing embryos compared to non-mated ones. Robust elevation of Adcyap1 and slight elevation of Hbegf were detected in females with blastocyst embryos compared with non-blastocysts. We found low rate of Hbegf mRNA expression in uncompacted embryos, whereas morulae and blastocysts expressed high amounts of Hbegf. However, we did not find detectable Adcyap1 mRNA in embryos. Strong correlation was found between uterine tissue and embryonic Hbegf levels, slight correlation between uterine Adcyap1 and Hbegf levels. Uterine tissue Adcyap1 and embryonic Hbegf showed no correlation. In summary, our present data show, for the first time, the correlation between PACAP and HB-EGF mRNA expression suggesting that PACAP might play a role during the peri-implantation period of early mouse embryo development.     

Oral–aboral axis specification in the sea urchin embryo,IV: Hypoxia radializes embryos by preventing the initial spatialization of nodal activity

The oral–aboral axis of the sea urchin embryo is specified conditionally via a regulated feedback circuit involving the signaling gene nodal and its antagonist lefty. In normal development nodal activity becomes localized to the prospective oral side of the blastula stage embryo, a process that requires lefty. In embryos of Strongylocentrotus purpuratus, a redox gradient established by asymmetrically distributed mitochondria provides an initial spatial input that positions the localized domain of nodal expression. This expression is perturbed by hypoxia, leading to development of radialized embryos lacking an oral–aboral axis. Here we show that this radialization is not caused by a failure to express nodal, but rather by a failure to localize nodal activity to one side of the embryo. This occurs even when embryos are removed from hypoxia at late cleavage stage when nodal is first expressed, indicating that the effect involves the initiation phase of nodal activity, rather than its positive feedback-driven amplification and maintenance. Quantitative fluorescence microscopy of MitoTracker Orange-labeled embryos expressing nodal-GFP reporter gene revealed that hypoxia abolishes the spatial correlation between mitochondrial distribution and nodal expression, suggesting that hypoxia eliminates the initial spatial bias in nodal activity normally established by the redox gradient. We propose that absent this bias, the initiation phase of nodal expression is spatially uniform, such that the ensuing Nodal-mediated community effect is not localized, and hence refractory to Lefty-mediated enforcement of localization.     

The evolution of porcine embryo in vitro production

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology.     

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

Embryo aberrations in the amphipod Monoporeia affinis as indicators of toxic pollutants in sediments: A field evaluation

Reproduction in the amphipod Monoporeia affinis is sensitive to contaminant exposure, and embryo aberrations are used in the Swedish National Monitoring Program to indicate biological effects of contaminants. However, empirical support for a causal relationship between contaminant load and frequency of aberrations is largely based on experimental studies. A field evaluation of aberration frequency in relation to contaminant exposure is required, if we are to use reproductive disorders as indicators of contaminant effects in risk assessment. In this study, we evaluated occurrence of females carrying malformed embryos and frequency of specific embryo aberrations in broods of M. affinis in relation to concentrations of various pollutants in sediments. Gravid M. affinis and surface sediment were sampled at ten sites in the Gulf of Bothnia located at varying proximity to historical and current polluting point sources. At each site, concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and trace and heavy metals in surface sediments were determined. Amphipod fecundity and embryo development status were assayed with regard to morphologically recognizable aberrations (malformed embryos, membrane-damaged embryos, embryos with arrested development and females with dead brood). Our key finding is that different types of embryo aberrations were significantly associated with specific contaminant groups in the sediment. In particular, occurrence of females with embryo malformations was strongly related to elevated concentrations of Cd and PCBs, while females with membrane-damaged embryos occurred at high PAH concentrations. Also, frequency of embryos with arrested development was linked to elevated concentrations of PAHs and metals. Finally, frequency of females carrying dead broods, which was earlier considered to be a hypoxia-induced pathology, was significantly positively related to PAH concentrations. Thus, these reproductive aberrations in M. affinis can serve as contaminant-specific indicators of PCB, PAH and heavy metal exposure in biological effect monitoring.     

Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT)

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated [1]. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission.     

Ovule morphology, embryogenesis and seed development in three Hylocereus species (Cactaceae)

Pre-embryonic and embryonic stages and seed developments were studied in the diploids Hylocereus monacanthus and Hylocereus undatus and the tetraploid Hylocereus megalanthus. Ovule morphology was similar among species except for micropyle entrance. H. monacanthus had the thickest and most robust suspensor. Embryo developmental time, measured from fertilization to maturity, was significantly more prolonged in H. megalanthus. Typical to Cactaceae, the seed coat was formed by one layer of sclerenchymatous cells, but was more lignified in H. megalanthus. Morphological features common to all species included (1) cellular type endosperm with independent patterns of development in the chalazal and micropylar zones, forming a haustorium layer from the chalazal zone to the embryo; (2) an endothelial layer surrounding the embryo sac almost complete; (3) a nucellar summit growing into the micropyle; and (4) a placental obturator and a funicle connecting the ovarian tissue to the ovule. Seed development was typically endospermic (exendospermic orthodox seeds). Anomalies included two egg cells in the same embryo sac, two embryos developing in the same ovule, and embryos developing from the chalazal pole region. Total seed number and seed viability were significantly lower in H. megalanthus than in the other two taxa. Embryos at different developmental stages were observed in aborted H. megalanthus seeds.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Prevention of bovine herpesvirus-1 transmission by the transfer of embryos disinfected with recombinant bovine trypsin

This study deals with the potential for the introduction of infectious agents through the use of animal-derived products. The efficacy of a recombinant bovine trypsin (RBTr) as a replacement for porcine pancreatic trypsin and a disinfectant for bovine herpesvirus-1 (BHV-1)–infected embryos was investigated according to the sanitary guidelines of the International Embryo Transfer Society. Treatment of in vivo and in vitro fertilized embryos contaminated with BHV-1 (10⁵ TCID50/mL) in the presence of RBTr (525 U/mL) for 120 s, effectively removed the infectious virus compared with untreated and washed embryos (P < 0.05). Transfer of in vivo fertilized and disinfected embryos to BHV-1 seronegative recipients (n = 24) resulted in 14 pregnancies and 11 calves born free of BHV-1. In contrast, transfer of unwashed or undisinfected embryos to four recipients resulted in seroconversion and no pregnancies at term. It was concluded that the use of RBTr could be considered as an alternative method of rendering embryos free of BHV-1 and thus reduce the potential risk of disease transmission to embryo recipients and offspring.     

Phosphatidylglycerol biosynthesis is required for the development of embryos and normal membrane structures of chloroplasts and mitochondria in Arabidopsis

Phosphatidylglycerophosphate (PGP) synthase, encoded by PGP1 and PGP2 in Arabidopsis, catalyzes a committed step in the biosynthesis of phosphatidylglycerol (PG). In this study, we isolated a pgp1pgp2 double mutant of Arabidopsis to study the function of PG. In this mutant, embryo development was delayed and the majority of seeds did not germinate. Thylakoid membranes did not develop in plastids, mitochondrial membrane structures were abnormal in the mutant embryos, and radiolabeling of phospholipids showed that radioactivity was not significantly incorporated into PG. These results demonstrated that PG biosynthesis is essential for the development of embryos and normal membrane structures of chloroplasts and mitochondria.     

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.     

In vitro production of small ruminant embryos: Late improvements and further research

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo–derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination.