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Abstract
This paper gives an overview of assisted reproductive technologies (ART) in livestock species coming from the author's direct experience and contribution to the development of several of them. The assessment is conducted on the basis of the progress achieved since the early eighties and the impact on the clinical/practical use of such procedures. Artificial insemination (AI) is still the leading technology used on a large scale in livestock with most favourable cost benefit ratio. All the other ARTs have niche applications compared to AI. Significant progress has been achieved in embryo culture, somatic cell nuclear transfer and on the identification of the many unknown variables affecting the success rate, while in areas such as superovulation, oocyte maturation, IVF, embryonic stem cells and cryopreservation progress has been limited or absent. It is the opinion of the author that ARTs have reached a plateau whereby only minimal improvement of efficiency can be achieved. Significant advances can only come from major breakthrough in the understanding of the underlying biological mechanisms.
... Созревание ооцита in vivo происходит при непосредственном участии структурных элементов фолликула, содержащихся в фолликулярной жидкости [3,4,6]. Клетки гранулезы широко используются в системах дозревания ооцитов коров, а также в технологиях клонирования и трансгенеза [8,13]. Цель настоящего исследования -проанализировать деструктивные изменения клеток гранулезы в овариальных фолликулах коров (Ø 3-5 мм), содержащих растущие (ВСВ -) или завершившие фазу роста ооциты (ВСВ + ). ...
... Этот процесс отвечает за развитие доминантного фолликула и желтого тела, фолликулярную атрезию и овуляцию [2,7]. Мониторинг деструктивных изменений гранулезных клеток овариальных фолликулов может явиться ключевым моментом в изучении механизмов формирования полноценной яйцеклетки [8,13]. ...
... Oocyte maturation in vivo occurs with the participation of structural follicle elements and follicular fluid [3,4,6]. Granulosa cells are widely used in bovine oocyte maturation systems and used in cloning and transgenesis technologies [8,13]. Purpose of this study: to perform destructive changes granulosa cells in ovarian follicles of bovines (Ø 3-5 mm),which contain growing(ВСВ -) or completed the growth phase of oocytes (ВСВ + ). ...
Currently, there is the possibility of more detailed studies to the study of oocytes and somatic cells (granulosa cells). The possibility to develop successful models of maturation of female gametes defines the possibility to improve existing methods for the selection of donor eggs and the search for new donor eggs. Oocyte maturation in vivo occurs with the participation of structural follicle elements and follicular fluid [3, 4, 6]. Granulosa cells are widely used in bovine oocyte maturation systems and used in cloning and transgenesis technologies [8, 13]. Purpose of this study: to perform destructive changes granulosa cells in ovarian follicles of bovines (Ø 3–5 mm),which contain growing(ВСВ–) or completed the growth phase of oocytes (ВСВ+). Methods: Functional testing of oocytes wascarried out using the vital dye BCB (brillant cresyl blue – diamond crystal blue) [10]. Viability indices in granulosa cells isolated from follicles that contain oocyte s that grow or complete the growth phase were determined by flow cytometry. The result. It was found, that cellsof granulosa from cow follicles are characterized by different indicators of apoptosis levels depending on the status of oocytes (completed growth and growing) isolated from these follicles. The proportion of apoptotic granulosa cells in bovine follicles of containing oocytes that completed the growth phase, exceeded that in follicles containing growing oocytes by 11 % (29 % vs. 18 %, c:dP < 0.05). The scientific novelty: The data obtained using flow cytometry, allow us to evaluate the level of apoptosis in granulosa cells of bovine ovarian follicle as indicator of functional status of developing oocytes (growing or completed growth phases). This indicator can be used in prognosis of competencies for maturation of bovine oocytes.
... Assisted reproductive technologies (ARTs) have come a long way in the last 30 years both in animals and humans, allowing in vitro embryo development until the blastocyst stage and successful pregnancy after embryo transfer [1]. In livestock, a substantial increase in the number of in vitro produced (IVP) embryos has been registered during the last years. ...
... Only 30% of cattle receiving IVP embryos will deliver a live calf, with 60% of these pregnancies failing during the first 6 weeks of gestation [2]. The persistent struggle is to maintain the pregnancy rate close to 50%, especially with IVP or cryopreserved embryos [1,2]. ...
Background
Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos.
Methods
For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing.
Results
In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01).
Conclusions
These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.
... Assisted reproductive technologies (ARTs) have become essential to managing reproduction in animal breeding, restoring species on the brink of extinction, and treating infertility in humans. However, the small supply of mature, fertilizable oocytes is one of the main limitations to the success rate of assisted reproduction (Lonergan & Fair 2008, Galli 2017. Even though the mammalian ovary is potentially the source of thousands of oocytes enclosed into follicles at various stages of development, only a few of them reach a stage ensuring the generation of viable embryos and can be successfully used for in vitro embryo production (IVEP). ...
The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3-D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus-oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-Ethynyl Uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes.
The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.
... In the past three decades, several strategies have been undertaken to overcome some of the intrinsic biological limits of these approaches. However, the percentage of success for in vitro embryo production (IVP) remained stunningly stable and approximately one-third of the oocytes isolated from a given ovary reach the blastocyst stage of embryonic development [1,2]. ...
In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies.
Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans.
An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.
... Despite the potential advances offered by in vitro embryo production (IVP) systems, the percentage of success in cow remained stunning stable over the last 30 years and is limited to one third of the oocytes isolated from the ovary reaching the blastocyst stage of embryonic development (Lonergan and Fair, 2008;Galli, 2017). In bovine IVP, on a percentage basis, starting from 100 oocytes only about one third become a blastocyst after IVM, IVF and IVC, and only about one third of these embryos are able to produce a born calf (Fig. 1). ...
The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques.
In the Republic of Kalmykia, the state of Kalmyk meat cattle is alarming, since proper selection work with the breed has not been carried out for many years, which led to a significant reduction in purebred livestock (Willett et al. in Science 113:247–248, 1951). The problem of the speedy restoration of breed qualities is extremely acute and its solution is possible only under the condition of accelerated reproduction of purebred genotyped livestock using embryo transplantation methods. Despite the fact that the embryotransfer method is currently in a position of functional self-sufficiency, some elements of the technology, such as stimulation of superovulation, are time-consuming or unpredictable, and cryopreservation methods carry high risks in relation to the preservation of the viability of embryos. The aim of the study is to develop effective ways to stimulate poliovulation and cryopreservation of embryos, as one of the fundamental factors in the embryotransfer methodology, in connection with the accelerated reproduction of the Kalmyk meat breed in nomadic animal husbandry. The work was carried out in 2010 in JSC “Kalmytskoye” for breeding work”, in 2017 in CJSC “Ababkovskoye” of the Nizhny Novgorod region and LLC “Batu plus” of Kalmykia and in 2022 in the Regional Research and Production Center for the Reproduction of Farm Animals of the FSBEI HE “Kalmyk State University named after B.B. Gorodovikov”. When developing methods of stimulation of poliovulation and cryopreservation of embryos, cows and heifers of mating age of Krasnogorbatovskaya meat and dairy and Kalmyk meat breeds were used. As a result of the research, a method for stimulating poliovulation with a prolonged form of FSH and a method for balancing embryos in a cryoprotector during freezing and thawing were developed.
The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte's differentiation.
In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.
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 the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), together with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: (i) dose dependent effects of cilostamide on nuclear maturation kinetics; (ii) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes; (iii) and the impact of treatments on developmental competence acquisition after parthenogenic activation (PA) and SCNT. Accordingly, COC were collected from 3-6 mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer Yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling up to 24 h of culture and delayed meiotic resumption as only 25.6% of cilostamide-treated oocytes reached ProMI stage compared to the control (69.7%; P<0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions, improved oocyte developmental competence as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.
Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
The only commercially viable method of sexing mammalian sperm is to use a flow cytometer to measure sperm DNA content via fluorescence of the DNA-bound fluorophore Hoechst 33342, and then sort sperm into three populations, probably X, probably Y, and undetermined. Millions of insemination doses of sexed sperm are produced annually by this procedure. Although accuracy of sexing usually exceeds 90%, this procedure of sexing one sperm at a time has serious limitations, including cost, sort rates, and damage to sperm resulting in lowered fertility, but not abnormalities in offspring. Suggested areas for research include determining how sperm are damaged and where in the process of fertilization and embryonic development the infertility is manifest. Pre and post sorting procedures are done in approximately hourly batches, and these might be changed to continuous procedures. Numerous genetic, physical, and immunological procedures for sexing millions of sperm in parallel have been proposed, but none appears to be suitable for commercialization at this time due to issues of accuracy, repeatability, damage to sperm, and other problems. However, increasing numbers of reports are appearing concerning improvements in these procedures, and it appears inevitable that one or more of them eventually will prove to be efficacious. In developing such procedures, it is critical to monitor sexing accuracy regularly by rapid and inexpensive procedures such as fluorescence in situ hybridization, quantitative PCR, or sort reanalysis by flow cytometry. Furthermore, monitoring fertility of sexed sperm such as in vitro fertilization should be integral to the development process. Intellectual property issues could be substantive.
During the 1970s, domestic animal biotechnology, i.e., embryo transfer in farm animals, was confronted with the problem of embryonic developmental arrest observed in vitro, especially during the cycle in which maternal to zygotic transition (MZT) cycle takes place. In farm animals, obtaining blastocysts is mandatory, as transfer at earlier stages results in expulsion of the embryo from the vagina. In humans, the first attempts to obtain blastocysts with classical culture media were disappointing, and the use of a coculture strategy was naturally tempting: the first significant results of successful blastocyst development were obtained in the early 1980s, using trophoblastic tissue as a feeder layer in order to mimic an autocrine embryotrophic system. The next supporting cell systems were based on oviduct epithelial cells and uterine cells in order to achieve a paracrine effect. Non-hormone dependence was then demonstrated with the use of prepubertal cells, and finally with the use of established cell lines of nongenital origin (African Green Monkey Kidney, Vero cells). The embryotrophic properties are linked to features of "transport epithelia." Vero cells have been extensively used in human ART, and most of our knowledge about the human blastocyst was gathered with the use of this technology. Coculture is still in current use, but with systems that employ autologous uterine cells. Results following the use of this technology in human ART are superior to those observed with the use of sequential media. The benefit is linked to the release of free radical scavengers and growth factors by the feeder cells. In animal biotechnology, an important part of the "precious embryos," i.e., those resulting from cloning technology, involves coculture with buffalo rat liver (BRL) cells or Vero cells.
As previously described for the establishment of stable, pluripotent cell lines from pig blastocysts, an analogous cell line was isolated from a sheep blastocyst. There are common features in the morphologies and growth characteristics of the pig and sheep cells in culture; in particular, pig and sheep cells display large nuclei and relatively sparse cytoplasm, as is observed in mouse embryonic stem cells. Furthermore, the morphology of the sheep cells closely resembles that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. This suggests that the sheep cell line represents a primary ectodermal lineage.
To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)
The aim of this study was to develop a serum-free culture system that could support high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. To this end, we investigated the effects on sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml). The inclusion of amino acids in the culture medium had no effect on the percentage of embryos arrested at the 8-16-cell stage when embryos were cultured singly in the same drop of medium for 6 days (43% in SOF; 41% in SOF+amino acids). However, in medium containing all Eagle's amino acids, replacing the culture medium every 48 h to alleviate ammonium toxicity significantly decreased the number of arrested embryos (6%; p < 0.05) and significantly increased blastocyst cell number (52 cells in SOF; 105 cells in SOF+amino acids; p < 0.01) and the number of embryos developing to the blastocyst stage (29% in SOF; 67% in SOF+amino acids; p < 0.05). When the medium was renewed every 48 h, nonessential amino acids and glutamine also significantly decreased the number of arrested embryos (p < 0.05). Culturing embryos singly or in groups in SOF medium with all Eagle's amino acids that was renewed every 48 h resulted in significant increases in blastocyst hatching and mean cell number (47%, 31%, and 79%; 105, 136, and 173 cells for embryos cultured singly, in groups of 2, and in groups of 4, respectively). After culture in groups of 4, blastocyst cell numbers were equivalent to in vivo-developed controls (160 cells) and significantly greater than those developed in serum (103 cells; p < 0.01). Analysis of blastocyst metabolism, expressed on a per-cell basis, revealed that amino acids did not affect either glucose uptake or lactate production, whereas the addition of amino acids and vitamins resulted in a significant increase in both parameters (p < 0.01). A similar response was observed in serum-derived blastocysts. Ammonium production by sheep blastocysts after culture in the presence of amino acids was significantly greater than that produced by mouse blastocysts, indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.(ABSTRACT TRUNCATED AT 400 WORDS)
Mammalian cloning using somatic cells has been accomplished successfully in several species, and its potential basic, clinical and therapeutic applications are being pursued on many fronts. Determining the long-term effects of cloning on offspring is crucial for consideration of future application of the technique. Although full-term development of animals cloned from adult somatic cells has been reported, problems in the resulting progeny indicate that the cloning procedure may not produce animals that are phenotypically identical to their cell donor. We used a mouse model to take advantage of its short generation time and lifespan. Here we report that the increased body weight of cloned B6C3F1 female mice reflects an increase of body fat in addition to a larger body size, and that these mice share many characteristics consistent with obesity. We also show that the obese phenotype is not transmitted to offspring generated by mating male and female cloned mice.
Does the manipulation of gametes and embryos as practised in human IVF invoke perturbations in fetal and neonatal phenotype?
There is increasing evidence that the answer is ‘yes’, although the degree of perturbation may be less acute than observed
in other species. However, the long-term consequences are not known, and may prove to be considerable. There is now a substantial
body of evidence from animal models suggesting that assisted reproductive technologies (ART) are associated with altered outcomes
in fetal and neonatal development. Epigenetic modification of gene expression is an attractive hypothesis that accounts for
these differences and is one of a number of causal pathways that may be activated by cellular stress invoked during manipulation.
Here we widen the debate to propose that environment-induced cellular stress also acts to modify fetal and placental gene
expression, potentially also contributing to phenotype skewing after ART.
IVF is limited by low success rates and an unacceptably high multiple pregnancy rate. These outcomes would be improved significantly if a single embryo of high viability could be replaced in each treatment cycle, but widespread acceptance of such a policy is hindered by the lack of predictive factors for embryo selection. We have conducted a retrospective clinical study of a novel non-invasive method of embryo selection based on the depletion/appearance of amino acids in the culture medium.
Fifty-three cycles of IVF treatment using ICSI were studied. Embryos were cultured for 24 h in 4 microl drops of medium containing a physiological mixture of 18 amino acids. The spent medium was analysed for amino acid content by high performance liquid chromatography.
The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical pregnancy and live birth. These correlations were independent of known predictors, such as female age, basal levels of FSH, embryo cell number and embryo morphological grade.
Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.
Concerns have been raised about the health and development of children conceived by assisted reproductive technologies (ART) since 1978. Controversially, ART has been linked with adverse obstetric and perinatal outcomes, an increased risk of birth defects, cancers, and growth and development disorders. Emerging evidence suggests that ART treatment may also predispose individuals to an increased risk of chronic ageing related diseases such as obesity, type 2 diabetes and cardiovascular disease. This review will summarize the available evidence on the short-term and long-term health outcomes of ART singletons, as multiple pregnancies after multiple embryos transfer, are associated with low birth weight and preterm delivery, which can separately increase risk of adverse postnatal outcomes, and impact long-term health. We will also examine the potential factors that may contribute to these health risks, and discuss underlying mechanisms, including epigenetic changes that may occur during the preimplantation period and reprogram development in utero, and adult health, later in life. Lastly, this review will consider the future directions with the view to optimize the long-term health of ART children.
Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex sorted product branded “SexedULTRA™” that retains sperm integrity to improve post thaw sperm quality, in vitro embryo production and field fertility compared with the previous XY method.
Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (Texas). Each ejaculate was divided in two aliquots and then processed in one of two methods (XY method or SexedULTRA™). Post thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3 h incubation at 37°C using a computer assisted sperm motility analyzer (Hamilton Thorne IVOS II system). Percent intact acrosomes (PIA) was also estimated after a 3 h incubation. Results were analyzed by a mixed model analysis of variance with the fixed effect of treatment and random effect of bull. Percent total motile SexedUltra™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 h (78.8% vs 67.2%) and 3 h (51.0% vs 39.0%) post thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 h (50.7% vs 44.9%) and 3 h (31.5% vs 4.4%) post thaw in the SexedULTRA™ sperm. PIA was also greater in SexedULTRA™ sperm compared to the sperm processed following previous method (78.0% vs 64.0%).
In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (Pennsylvania). 5-10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of three straws and a minimum of 800 oocytes per treatment group (ejaculate x treatment) were included in the comparison for development to 8 cell stage (cleavage rate) and to day 7 blastocyst stage measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analyzed using a mixed model analysis of variance with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P<0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex sorting process results in better semen quality post thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex sorted bovine sperm.
During the last two decades, research on in vitro preantral follicle growth and oocyte maturation has delivered fascinating advances concerning the knowledge of processes regulating follicle growth and the developmental competence of oocytes. These advances include: (1) information about the role of several hormones and growth factors on in vitro activation of primordial follicles; (2) increased understanding of the intracellular pathway involved in the initiation of primordial follicle growth, (3) the growth of primary and secondary follicles up to antral stages, and (4) production of embryos from oocytes from in vitro grown preantral follicles. This review paper describes these advances, especially in regard farm animals, and discusses the reasons that limit embryo production from oocytes derived from preantral follicles cultured in vitro.
For heifers, beef and moderate-yielding dairy cows, it appears that the fertilisation rate generally lies between 90% and 100%. For high-producing dairy cows, there is a less substantive body of literature, but it would appear that the fertilisation rate is somewhat lower and possibly more variable. In cattle, the major component of embryo loss occurs in the first 16 days following breeding (Day 0), with emerging evidence of greater losses before Day 8 in high-producing dairy cows. In cattle, late embryo mortality causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate. The application of direct progesterone supplementation or treatments to increase endogenous output of progesterone to increase embryo survival cannot be recommended at this time. Energy balance and dry matter intake during the first 4 weeks after calving are critically important in determining pregnancies per AI when cows are inseminated at 70-100 days after calving. Level of concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates, although sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy must be developed. There is genetic variability within the Holstein breed for fertility traits, which can be exploited. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. In addition, there is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.
Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.
In this preliminary note I wish merely to record an experiment by which it is shown that it is possible to make use of the uterus of one variety of rabbit as a medium for the growth and complete fœtal development of fertilised ova of another variety of rabbit. Briefly, the experiment made was as follows:—On the 27th April, 1890, two ova were obtained from an Angora doe rabbit which had been fertilised by an Angora buck thirty-two hours previously; the ova were undergoing segmentation, being divided into four segments.
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The scientific identification of the key components of sexual reproduction – eggs and sperm – took place during an amazing decade of discovery in the 1660s and 1670s. The names of many of the people involved are now forgotten, and yet their work, and the difficulties they faced and the conflicts they endured, resonate strongly to the present day. Despite this period of innovation, the respective roles of egg and sperm remained unclear for another 170 years. Why did this take so long? And what did people think before these discoveries? By tracing the contours of this major milestone in human knowledge, we can also gain insight into our current knowledge, and the boundaries we may be unwittingly trapped by.
The hypothesis that follicle cells play a central role in endowing the oocyte with developmental competence during maturation has been tested in coculture studies. Oocytes and follicle cells in various combinations were cultured for 24 h with gonadotrophins and estrogen in a nonstatic culture system. Developmental competence was assessed by transfer of oocytes to inseminated receipient ewes followed by embryonic examination 12 days later or at parturition.
Denuded and corona-enclosed oocytes resume meiosis in culture but remain immature and developmentally incompetent. By contrast, oocytes supported by the cumulus and underlying granulosa (cumulus-oocyte complexes) undergo full maturation and normal subsequent embryonic development (42.6% to embryos). Addition of supplementary follicle cells during culture (5 × 106 cells/ml medium) is without beneficial effect on denuded oocytes. However, supplementary cells confer competence on corona-enclosed oocytes (37% to embryos).
A dual cellular requirement for full maturation involving both cell numbers and some direct cell-oocyte contact is highlighted by our experiments. The results demonstrate further that the nonstatic system provides a simple but reliable method of producing large numbers of fully matured oocytes for both research and clinical purposes.
In the buffalo, the use of embryo-based biotechnologies for breeding and genetic improvement is still very limited because multiple-ovulation embryo transfer delivers poor results compared with cattle and in vitro embryo production has been used mainly for research purposes. At present, very few reports are available on the transfer of in vitro-produced (IVP) and cryopreserved buffalo embryos. Therefore, the scope of this work was to perform a pilot study to evaluate the viability of frozen-thawed IVP embryos by nonsurgical embryo transfer to recipients in an IVF-embryo transfer program on a farm located on the north coast of Colombia, South America. Buffalo oocytes were recovered at the slaughterhouse from selected donors, matured in vitro for 18 to 20h in TCM-199+10% FCS and 0.5 IU of FSH and 0.5 IU of LH in 5% CO(2) at 38.5°C. Four different bulls were used for IVF. After thawing, the semen was separated on a Percoll(®) gradient and then diluted into SOF-IVF media supplemented with 1μgmL(-1) of heparin and phenylalanine. Presumptive zygotes were cultured in modified SOF supplemented with MEM amino acids for 6 days. Half of the medium was replaced on Day 4 and 6. Developing embryos were selected for freezing on Day 6 and 7. Grade 1 embryos were frozen at the blastocyst stage by slow cooling in 10% glycerol or 1.5M ethylene glycol. Recipients (heifers n=79 and uniparous cows n=17) were synchronized using the CIDR-Synch protocol: on Day 0, gonadotropin-releasing hormone was injected and a CIDR was inserted; on Day 7, prostaglandin F(2α) was administered; on Day 9, the CIDR was removed; on Day 11, a second injection of gonadotropin-releasing hormone was given; and on Day 17, the embryo was transferred. Each female received, nonsurgically, 1 or 2 embryos in the ipsilateral horn to the functional corpus luteum evaluated by ultrasonography. Pregnancies were evaluated by ultrasonography 30 days after transfer and confirmed by rectal palpation 30 days later. This work was performed in 2 successive experiments during the breeding seasons (January and December, respectively). Overall, 96 recipients were transferred, with 136 embryos obtaining 23 pregnancies (24.2%). There were no statistical differences in pregnancy rate between heifers and cows (25.3 vs 17.7%) and between single (n=56) and double (n=39) embryo transfers (21.4 vs 27.5%) by chi square test (P>0.05). To date, 4 females and 5 males have been born by spontaneous calving (1 stillborn male due to dystocia), 3 pregnancies have been aborted (13%) and 11 pregnancies are ongoing (>7 months). The pregnancy rate obtained in this study in farm conditions (24.2%) is lower than generally obtained with frozen IVP cattle embryos, but it is still a good result in buffalo, where even conventional AI provides a lower success rate as compared with cattle. Finally, this work demonstrates that in vitro embryo production can be successfully implemented in buffalo breeding programs for the exploitation of superior genetics.
Day 7 cow embryos were frozen in 1.5 M-DMSO in PBS at 0.3 degrees C/min to -36 degrees C and at 0.1 degrees C/min between -36 and -60 degrees C before being plunged directly into liquid nitrogen. They were subsequently thawed (rapidly to -50 degrees C, at 4 degrees C/min from -50 to -10 degrees C, and rapidly again) to room temperature. Embryonic viability was tested by four different transfer techniques. Maximum pregnancy rate (8/12) was obtained with surgical transfer immediately after thawing and dilution of DMSO.
Intracytoplasmic sperm injection (ICSI) is a promising assisted-fertilisation technique that may benefit women who have not become pregnant by in-vitro fertilisation (IVF) or subzonal insemination (SUZI) of oocytes. We have used ICSI to treat couples with infertility because of severely impaired sperm characteristics, and in whom IVF and SUZI had failed. Direct injection of a single spermatozoon into the ooplasm was done in 47 metaphase-II oocytes: 38 oocytes remained intact after injection, 31 became fertilised, and 15 embryos were replaced in utero. Four pregnancies occurred after eight treatment cycles--two singleton and one twin pregnancy, and a preclinical abortion. Two healthy boys have been delivered from the singleton pregnancies and a healthy boy and girl from the twin pregnancy.
Since the first successful collection of oocytes by non-surgical puncture, there have been numerous attempts to fertilize them but few segmented embryos have resulted. The latest attempts at follicular puncture (Palmer et al., 1987) provided 159 oocytes. Oocytes found broken (18%) were probably already broken, or at least fragile, before puncture. The 41 oocytes were fertilized only with semen treated with Ionophore A23187. Following ionophore treatment of semen, 16 ova segmented (of 113 inseminated oocytes) indicating fertilization, and another 7 showed signs of fertilization but not segmentation. Our basic protocol, when applied to 60 oocytes, yielded 11 (18%) embryos and 5 (8%) that were fertilized without segmentation. Modifications to the protocol produced no improvement in the results. Eight embryos fertilized in vitro were transferred into the ampulla of 8 recipient mares. One pregnancy resulted and foaling occurred June 14, 1990. This is the first equine pregnancy to continue to full term following in vitro fertilization.
Important interactions occur between the somatic cells of the oviduct and the developing embryo, during the later stages of pre-attachment development. In these stages secretions from the oviduct are very important in conferring viability on the embryos, especially plasmatic proteins and proteins secreted by the oviduct cells. The oviduct secretions obtained at various stages of the oestrus cycle have now been tested for potential growth factors. If an active fraction of the oviduct secretion is identified, the potential embryotrophic factor(s) will be characterized and produced on a large scale so that culture medium can be supplemented.
The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.
A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.
In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.
Embryonic stem cell technology is now well established in the mouse (reviewed by Robertson, 1987). This technology implies the isolation from the preimplantation embrao of a cell line (ES) that is cultured in vitro in an undifferentiated state. Embryonal carcinoma cells (EC) lines obtained from malignant tumours (Martin, 1975), together with all the information available on their culture requirements (reviewed by Heath, 1987), represented a very important starting point for the establishment of ES cells (Martin, 1981). ES cells share many characteristics with EC cells such as the ability to contribute to somatic tissues of animals obtained following injection of cells into a host blastocyst, to differentiate in vitro under appropriate stimuli (Rudnicki & McBurney, 1987) and to form retransplantable tumours. ES cells, however, have substantial advantages over EC cells in that they can be derived directly from a normal embryo, they maintain a normal karyotype and when reintroduced into a host blastocyst they can colonise the germ line (Bradley, 1987). ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent to the cells from differentiating. It has been shown that glycoproteins are responsible for this effect and these have been named according to their different activities: DIA, differentiation inhibitory activity (Smith & Hooper, 1987); LIF, leukaemia inhibiting factor (Smith et al, 1988; Williams et al, 1988); HILDA, human interleukin for DA cells (Moreau et al., 1988). It is now possible to establish and maintain ES cells in culture in the absence of feeders cells but in the presence of such factors (Nichols et al., 1990).
The objective of these studies was to achieve complete oocyte development in vitro beginning with the oocytes in the primordial follicles of newborn mouse ovaries. A two-step strategy was developed: first the ovaries of newborn mice were grown in organ culture for 8 days, and then the developing oocyte-granulosa cell complexes were isolated from the organ-cultured ovaries and cultured for an additional 14 days. The oocytes of primordial follicles are approximately 4190 microns3 in volume (20 microns in diameter), and this volume increased by approximately 53,810 microns3 to a final size of 58,000 microns3--a 13.8-fold increase--during the 8 days of organ culture. In the first experiment the oocyte-granulosa cell complexes were grown in control medium or in medium supplemented with FSH (0.5 ng/ml), epidermal growth factor (EGF; 1.0 ng/ml), or EGF plus FSH. Only 50-60% of the complexes cultured in control medium or in medium supplemented with FSH were recovered at the end of the 14-day culture period. In contrast, more than 90% of the complexes cultured in medium supplemented with EGF were recovered. The median size of the oocytes grown in control medium was 176,800 microns3 (69-microns diameter), while the median size of those grown in medium supplemented with EGF was slightly smaller (136,400-microns3 volume; 63-microns diameter), due to the survival of more smaller-size oocytes in EGF-containing medium. Thirty percent of the oocytes recovered after development in FSH-containing medium were competent to undergo germinal vesicle breakdown (GVB). In the second set of experiments, oocyte-granulosa cell complexes isolated from organ-cultured ovaries were cultured in medium supplemented with either 0.5 or 5.0 ng/ml FSH or with these same concentrations of FSH plus 1.0 ng/ml EGF. Again, increased oocyte recovery was observed in the groups cultured with EGF. There was no difference among the groups in the percentage of the oocytes that acquired competence to undergo GVB (32%) or in the percentage of GVB oocytes that produced a polar body, thus indicating progression of meiosis to metaphase II (22%). When the mature oocytes were inseminated, 21% underwent fertilization and cleavage to the 2-cell stage in the groups without EGF during oocyte development, while 42% underwent fertilization and cleavage to the 2-cell stage in the groups cultured with EGF. Less than 2% of the 2-cell-stage embryos developed to the blastocyst stage in any of the groups. One hundred and ninety 2-cell-stage embryos were transferred to the oviducts of pseudopregnant females; two females produced one pup each; one was living and the other had apparently died recently. The results reported here clearly show that complete development of oocytes in vitro from the primordial follicle stage is possible and establish the framework for further studies using oocytes from laboratory animals as model systems for the development of oocytes from humans as well as from animals of agricultural and zoological importance.
Expression of various developmentally regulated markers was screened throughout the preimplantation stages of in vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as F-actin, alpha-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine embryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells of in vitro-derived bovine embryos valid for cell line derivation.
Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipient's oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.
Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique. It can be performed twice a week for many weeks without side effects on the donor's reproductive career. The donor can be in almost any physiological status and still be suitable for oocyte recovery. A scanner with a sectorial or convex probe and a vacuum pump are required. Collection is performed with minimal stress to the donor. An average of 8 to 10 oocytes are collected per OPU with an average production of 2 transferable embryos. The laboratory production of embryos from such oocytes does not differ from that of oocytes harvested at slaughter as the results after transfer to final recipients. For other species such as buffalo and horses OPU has been attempted similarly to cattle and data will be presented and reviewed. For small ruminants, laparotomy or laparoscopy seems the only reliable route so far to collect oocytes from live donors.
Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.
The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.
Cloned animals suffer a wide range of severe fetal and placental malformations. Whether these malformations arise from insufficient epigenetic modifications or mutations has not yet been determined. To address this question, we examined siblings from both cloned XO and XY parents. These parents, which exhibited hypertrophic placentas, increased body weights, and open eyelids at birth, were created from the same ES cell sublines. The siblings from all three cloned pairs showed normal body and placenta weights and no open eyelids at birth. The results clearly showed that the phenotypic abnormalities seen in cloned mice were not transmitted to the progeny, a finding that suggests that abnormalities in cloned mice are responsible for insufficient epigenetic modifications/reprogramming.
The history of research into artificial insemination (AI) is over two centuries old and its commercial application now spans 75 years. It is appropriate to reflect on the contribution of this powerful method of gene dispersal. AI remains as one of the most important assisted reproductive technologies. The three cornerstones for its application are: it is simple, economical and successful. The importance of AI will be challenged in the next few decades. The remarkable progress made in other assisted reproductive technologies does have the potential to rapidly generate offspring. The challenge for any of these reproductive technologies to attain widespread use is to match AI in being simple, economical and successful. This review aims at capturing the salient advances in AI, the comparisons with natural mating and other reproductive technologies, and, whether the future of AI will be challenged. It predicts what the new horizon looks like and the role that AI will play in the overall reproductive technologies landscape.
Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.
The development of bovine embryos obtained by intracytoplasmic sperm injection (ICSI) was studied in relation to various treatments applied to the sperm and to the early embryo. We investigated the effect of different activation protocols on ICSI-embryos and the influence of sperm capacitation with heparin and D-penicillamine, hypotaurine, and epinephrine (PHE) prior to ICSI. Finally, we studied the effect of dithiothreitol (DTT) pre-treatment of sperm or of injected oocytes. The activation of ICSI-embryos by ionomycin (Io)-cycloheximide (CHX) and sperm pre-treatment with heparin in combination with PHE did not increase the developmental capacity of ICSI-embryos. By contrast, the treatment of injected oocytes with 2 mM DTT resulted in increased cleavage and blastocyst rates in the group of non-activated embryos and in acceleration of blastocyst development in the group of activated embryos. Similarly, pre-treatment of sperm with DTT, followed by ICSI and activation, determined an increase of embryo development on Day 7 although the total number of blastocysts recorded on Day 8 was not different from untreated controls. The transfer of 11 ICSI-blastocysts, produced without activation, in six recipients gave rise to two pregnancies of which one went to term with the birth of an healthy calf.
Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor's genetic material to the offspring of recipient animals. Germ cell transplantation provides a bioassay to study the biology of male germ line stem cells, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis and treat male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals. In domestic animals including pigs, goats and cattle, as well as in primates, germ cells can be transplanted to a recipient testis by ultrasonographic-guided cannulation of the rete testis. Germ cell transplantation was successful between unrelated, immuno-competent pigs and goats, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in the production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in domestic animal species where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including horses and primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in domestic animals.
We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.