Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Gametes alter the oviductal secretory proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.     

Transcriptome Profiling of Embryonic Development Rate in Rainbow Trout Advanced Backcross Introgression Lines

In rainbow trout (Oncorhynchus mykiss) and other fishes, embryonic development rate is an ecologically and evolutionarily important trait that is closely associated with survival and physiological performance later in life. To identify genes differentially regulated in fast and slow-developing embryos of rainbow trout, we examined gene expression across developmental time points in rainbow trout embryos possessing alleles linked to a major quantitative trait loci (QTL) for fast versus slow embryonic development rate. Whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family, whereby each backcross generation involved the introgression of the fast-developing alleles for a major development rate QTL into a slow-developing clonal line of rainbow trout. Embryos were collected at 15, 19, and 28 days post-fertilization; sex and QTL genotype were determined using molecular markers, and cDNA from 48 embryos were used for microarray analysis. A total of 183 features were identified with significant differences between embryonic development rate genotypes. Genes associated with cell cycle growth, muscle contraction and protein synthesis were expressed significantly higher in embryos with the fast-developing allele (Clearwater) than those with the slow-developing allele (Oregon State University), which may associate with fast growth and early body mass construction in embryo development. Across time points, individuals with the fast-developing QTL allele appeared to have earlier onset of these developmental processes when compared to individuals with the slow development alleles, even as early as 15 days post-fertilization. Differentially expressed candidate genes chosen for linkage mapping were localized primarily to regions outside of the major embryonic development rate QTL, with the exception of a single gene (very low-density lipoprotein receptor precursor).     

The effect of acidification on oogenesis of rainbow trout during sex differentiation

Rainbow trout larvae were kept in acid water from 16 to 55 days after hatching. Initially, the acid environment stimulated a significant increase of gametes in the gonads of females at all developmental stages, but after longer exposure the stimulation was replaced by inhibition of gonadal development in the treated fish. In some fish, gamete reserves increased, while in others there was increased growth and maturation of late-generation oocytes.     

Failure of interspecies androgenesis in salmonids

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis . Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss ), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar ) and Salvelinus (brook trout and Arctic charr, S. alpinus ). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.     

Gamete origin in relation to early embryo development

Fertilization in vivo requires a complex series of selection events to occur in order to guarantee that only the fittest gametes take part in the fusion process and give rise to a viable embryo. Conventional practice in bovine in vitro fertilization however is to select oocytes and sperm by quite crude procedures. It is therefore not inconceivable that essentially unfit gametes may drive aberrant embryo development in vitro. Abnormal embryonic cells are being removed by apoptosis, which is a physiological process in embryos. Only an excess or a lack of apoptosis can lead to embryonic death or abnormal development. Suboptimal culture conditions undoubtedly contribute to undue embryonic apoptosis, but the intrinsic quality of the oocyte may also be a causative factor. It is generally accepted that the oocyte is in control of early embryogenesis, but is it also in control of future embryonic suicide? Is a compromised follicular environment predestining the oocyte to a dire fate? What is the contribution of the cumulus cells to oocyte quality, and can they rescue it from early demise? And what can be said about the origin of the spermatozoa? Research in human in vitro fertilization has definitely shown that factors such as paternal age, smoking and other sperm stressors can contribute to abnormal embryo development and even diseased offspring. This review will address the questions raised above, and will describe what is known about the cellular and molecular biology that may account for abnormal bovine embryo development caused by gamete origin.     

Production of F0 mice from embryonic stem cells injected eight-cell stage embryos which stored at refrigeration temperature

At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Semen from rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreservation

Oocytes from three female rainbow trout Oncorhynchus mykiss were inseminated separately with untreated or cryopreserved semen, which had been produced using either untreated (three males) or cryopreserved (three males) spermatozoa. In half of variants, the cryopreservation did not significantly affect fertilization efficiency. Regardless of whether the sperm donors were produced from cryopreserved or intact semen, insemination of oocytes with their intact sperm resulted in the same percentage of eyed embryos (94.4 and 94.3%, respectively). When eggs were inseminated with cryopreserved semen, the use of sperm from males produced with cryopreserved spermatozoa resulted in a significantly higher percentage of eyed eggs than in case of donors produced with intact sperm (89.6 and 81.7%, respectively). The production of rainbow trout using cryopreserved sperm does not appear to negatively affect reproductive abilities of male progeny and semen from donors, which were produced using cryopreserved sperm, is more suitable for cryopreservation than the semen from donors produced with intactspermatozoa.     

Refrigerated storage of European common frog Rana temporaria oocytes

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1–5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1–5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.     

Fundamental cryobiology of reproductive cells and tissues

During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor-recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen-thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species' sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species' embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species' oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.     

Chromatin in early mammalian embryos: achieving the pluripotent state

Abstract Gametes of both sexes (sperm and oocyte) are highly specialized and differentiated but within a very short time period post-fertilization the embryonic genome, produced by the combination of the two highly specialized parental genomes, is completely converted into a totipotent state. As a result, the one-cell-stage embryo can give rise to all cell types of all three embryonic layers, including the gametes. Thus, it is evident that extensive and efficient reprogramming steps occur soon after fertilization and also probably during early embryogenesis to reverse completely the differentiated state of the gamete and to achieve toti- or later on pluripotency of embryonic cells. However, after the embryo reaches the blastocyst stage, the first two distinct cell lineages can be clearly distinguished—the trophectoderm and the inner cells mass. The de-differentiation of gametes after fertilization, as well as the differentiation that is associated with the formation of blastocysts, are accompanied by changes in the state and properties of chromatin in individual embryonic nuclei at both the whole genome level as well as at the level of individual genes. In this contribution, we focus mainly on those events that take place soon after fertilization and during early embryogenesis in mammals. We will discuss the changes in DNA methylation and covalent histone modifications that were shown to be highly dynamic during this period; moreover, it has also been documented that abnormalities in these processes have a devastating impact on the developmental ability of embryos. Special attention will be paid to somatic cell nuclear transfer as it has been shown that the aberrant and inefficient reprogramming may be responsible for compromised development of cloned embryos.     

The space-time distribution of the mRNA of the nuclear proteins c-myc and P-53 in the development of the clawed toad studied by hybridization in situ]

We studied distribution of mRNA for nuclear protooncogene c-myc and nuclear protein P-53 in mature oocytes and embryos of Xenopus laevis from the stage of fertilization up to the stage of hatching by in situ hybridization with histological sections. mRNA for c-myc was present in all cells of the embryo at all studied developmental stages. Between the stage of fertilization and up to the late blastula, mRNA concentration for c-myc decreased progressively in all embryonic cells. During gastrulation a local increase in the concentration of this messenger was found in dorsal mesoderm and ectoderm. At the stage of neurula increased concentration of mRNA for c-myc was observed in all cells of the embryo but the hybridization signal increased particularly distinctly in cells of the neural tube. In studies of P-53 mRNA distribution hybridization signal was detected only in brain cells after stage 20 of development (after closure of the neural folds) and up to the stage of hatching.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.     

Influence of the duration of gamete interaction on cleavage,growth rate and sex distribution of in vitro produced bovine embryos

Various factors including the length of gamete interaction and embryo culture conditions are known to influence the rate of development and sex ratio of mammalian embryos produced in vitro. While the duration of gamete interaction deemed optimum would vary depending upon the species involved and the preferred sex in the outcome of in vitro procedures, the mechanisms favoring the selection of embryos of one sex over the other, or the exact time of post-fertilization stage at which a sex-related difference in growth rate is manifested, are not fully understood. In order to determine the optimum length of gamete co-incubation and the impact of male gamete 'aging' on the growth rate and sex ratio of bovine embryos, a series of experiments was carried out using in vitro matured (IVM) oocytes. In experiment 1, IVM oocytes were co-incubated with sperm from two different bulls for 6, 9, 12 and 18 h and the presumptive zygotes were cultured for approximately 7.5 days (178-180 h post-insemination (hpi)) prior to assessing the cleavage rate, blastocyst yield and the sex ratio of blastocysts in each co-incubation group. In experiment 2, the blastocysts obtained from different co-incubation groups were subjected to differential staining to determine the total cell number (TCN) and the proportion of cells allocated to the inner cell mass (ICM) in male and female embryos to test for sex-related differences in cell proliferation or in differentiation of the two embryonic cell lineages in the blastocysts. In experiment 3, IVM oocytes co-incubated for 6, 9, 12 and 18 h with sperm from a single bull, were cultured for 3 days (72 hpi) and the pre-morulae, categorized according to the specific stage of early development, were sexed to determine if a sex-dependent difference is detectable before the blastocyst stage. In experiment 4, IVM oocytes exposed to prolonged co-incubation (18 and 24 h) were compared with those co-incubated with "aged" (pre-incubated) sperm to determine if "aging sperm" is a factor affecting the growth rate and sex ratio of the out come. Our experiments showed that (1) the shortest period (6 h) allowed the highest proportion of cleaved oocytes to reach the blastocyst stage regardless of the semen donor, (2) males out number females (over 2 to 1) among blastocysts when co-incubation of gametes is reduced to 6 h, (3) the male blastocysts display higher total cell count, and (4) the faster growth rate of the male embryos does not affect the early differentiation and allocation of cells to the ICM. Furthermore, our results indicate that the disruption of the expected 1:1 ratio for male and female embryos in the short term co-incubation group is evident as early as the 4-cell stage and peaks at the 8-cell stage and that prolonged gamete interaction tends to reduce the blastocyst yield to even out the sex ratio. Absence of a significant effect on the yield and sex ratio of blastocysts in the prolonged co-incubation groups irrespective of the type of sperm (aged versus non-aged) used suggest that the preponderance of male embryos in short term gamete interaction group may be dependent upon the in vitro advantage of the Y-chromosome bearing sperm. This advantage, manifested in the precocious development during the pre-morulae stage is confined to a short duration that is neutralized when gamete interaction is allowed to proceed beyond 6h.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.     

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being "movers" to "fertilizers." Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization.     

Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death

To develop a model for utilizing germ cells collected from dead animals, male mice were euthanized and refrigerated for various periods, and the viability of the epididymal spermatozoa was examined by in vitro fertilization, embryo culture, and embryo transfer. Higher proportions of fresh oocytes were fertilized when males had been stored at 4-6 or 8-10 degrees C than at 0 degrees C. By partially dissecting the zona of freshly ovulated oocytes, spermatozoa from ICR male mice could fertilize oocytes (21% fertilization rate) after being stored for 5 days at 4-6 degrees C, and spermatozoa from BDF1 male mice could fertilize oocytes (39%) after being stored for 7 days at 4-6 degrees C. The resulting two-cell embryos had the ability to develop into expanded blastocysts in culture (81-100%) and into live young after transfer (34-47%). With further refinement of this system, it should be applicable not only for rescuing valuable genetic variants in laboratory animals or livestock animals but also for wild species in the future.     

Animal oocyte and embryo cryopreservation

Cryopreservation of oocytes and embryos is a crucial step for the widespread and conservation of animal genetic resources. However, oocytes and early embryos are very sensitive to chilling and cryopreservation and although new advances have been achieved in the past few years the perfect protocol has not yet been established. All oocytes and embryos suffer considerable morphological and functional damage during cryopreservation but the extent of the injury as well as differences in survival and developmental rates may be highly variable depending on the species, developmental stage and origin (for example, in vitro produced or in vivo derived, micromanipulated or not). Currently, there are two methods for gamete and embryos cryopreservation: slow freezing and vitrification. We have experienced both techniques but vitrification has become a viable and promising alternative to traditional approaches especially when dealing with in vitro produced or micromanipulated embryos and oocytes. Recently new strategies based on emerging studies in the field of lipid research have been used to reduce intracellular lipid content in bovine in vitro produced embryos and therefore increase their tolerance to micromanipulation and cryopreservation. The addition of a conjugated isomer of linoleic acid, the trans-10, cis-12 octadecadienoic acid to embryo culture medium more than twice improved embryo post-thawing viability after micromanipulation and vitrification. Vitrification was also used for the cryopreservation of embryos belonging to the Portuguese Animal Germplasm Bank project presently running at our facilities. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 2007 at Vale de Santarém, Portugal.