Recipient Embryos Research Articles

Effects of a reusable progesterone device on conception rates and estrus cycle re-synchronization in Nelore cows

The objective of this study was to evaluate the reuse of an intravaginal progesterone (P4) device during diestrus of Nelore cow embryo recipients in order to improve conception rate, accelerate estrus cycle re-synchronization of non-pregnant animals, and reduce the amount of waste generated by animal breeding biotechnologies. Two experiments were performed using 268 multiparous Nelore cows. In experiment 1, all animals were subjected to a timed embryo transfer (TET) procedure, but at the time of embryo transfer, two treatment groups were established: T1 - the control treatment (N = 132) and T2 - animals receiving a second-use CIDR® device for 12 days (N = 136). Experiment 2 was performed on cows that had not remained pregnant after experiment 1 using two groups: G1 - a control group (N = 69) and G2 -re-synchronized cows that received a P4 device for 12 days for the first TET (N = 74). In experiment 1, no significant effect of the P4 treatment was observed on conception rate (T1 = 37.9%, T2 = 39.7%; P = 0.50) and corpus luteum (CL) diameter (T1 = 17.5 ± 3.4 mm, T2 = 18.1 ± 3.4 mm; P = 0.61). In experiment 2, no significant effect of the treatment was observed on conception rate (G1 = 22.2%, G2 = 35.7%; P = 0.24), recipients utilization rate (G1 = 75.4%, G2 = 70.3%; P = 0.86), and CL diameter (G1 = 17.4 ± 3 mm, G2 = 18.1 ± 3.2 mm; P = 0.27). However, the P4 treatment (for re-synchronization) significantly increased the conception rate (G1 = 22.2%, G2 = 35.7%; P = 0.04), which was similar to that in a conventional TET protocol performed with a new device (38.8%). We conclude that reusable intravaginal P4 devices can help accelerate TET protocols, suggesting an alternative application method; furthermore, this protocol may help reduce waste production in assisted animal breeding.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane’s rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

Attempts to obtain fully xenogeneic fetuses in rat ↔ mouse model†,‡

The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.

Use of color-Doppler ultrasonography for selection of recipients in timed-embryo transfer programs in beef cattle

We aimed to study the association between CL characteristics assessed by color-Doppler ultrasonography (Doppler-US) at the time of embryo transfer (ET) and pregnancy rate (P/ET) in beef recipients. Estrous cycles of crossbred beef recipients were synchronized for timed-ET. On the day of ET (Day 7), CL area, proportion of luteal blood perfusion (BP), and the relationship between the largest dominant follicle (DF) and CL (ipsilateral or contralateral) were determined. Animals (n = 444) received an in vitro produced embryo from Nelore donors, placed in the uterine horn ipsilateral to the CL. Recipients were split retrospectively in three subgroups according to CL area [small (<3 cm2), medium (3–4 cm2), or large (>4 cm2)] and three subgroups according to luteal signals of BP [low (≤40%), medium (45–50%) or high (≥55%)]. Pregnancy was detected on Days 30–45 by transrectal ultrasonography and P/ET was analyzed considering the effects of cow's category (suckling or non-suckling), CL area, luteal BP and side of DF. P/ET increased along with BP category [low, 45.9%B, (62/135); medium, 54.1%AB (93/172); and high, 58.4%A (80/137)]. When luteal BP was evaluated as a continuous variable, a significant (P < 0.05) linear and positive effect was observed on P/ET. A greater (P < 0.05) CL area and serum progesterone concentrations were observed in the medium and high BP than in the low BP category. Although an effect of luteal size category was not significant on P/ET [small, 49% (76/155); medium, 59.7% (83/139); and large, 50.7% (75/148); P > 0.1], when CL area was evaluated as a continuous variable, a quadratic effect (P < 0.05) indicated a positive relationship between P/ET and CL area until luteal tissue reached 4.07 cm2, followed by a negative relationship. The location of the first-wave DF in relation to the CL did not affect P/ET (P > 0.1). In conclusion, Doppler-US is an innovative tool that has the potential to be used for selection of suitable embryo recipients based on luteal BP. Selection of recipients that have a greater chance of maintaining pregnancy will increase the success of timed-ET programs.

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†.

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

In vitro culture and characterization of duck primordial germ cells

ABSTRACTThis study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Effects of supplemental progesterone using a CIDR insert on pregnancy per embryo transfer of dairy heifer recipients of embryos produced in vitro

The objective of this experiment was to evaluate effects of supplemental progesterone immediately following transfer of frozen-thawed, IVP embryos on P/ET. Holstein heifers (n = 452), allocated to nine transfer groups over time, were assigned to be embryo recipients in a completely randomized study from December 2016 to April 2017. All heifers were randomly assigned to one of two treatments: 1) control (CON; n = 212) with no further treatment, or 2) received a CIDR insert containing progesterone for 12 d, beginning on the day of transfer (D 7) and removed 12 d later on Day 19 (CIDR; n = 228). A subset of heifers were subjected to blood sampling on Day 7 (ET) and Day 19 (CIDR removal) to determine circulating concentrations of progesterone. Pregnancy was initially determined using a serum assay for pregnancy specific protein-B at approximately Day 40 after ET and confirmed a month later using trans-rectal ultrasonography. Overall, P/ET did not differ (P = 0.941) between treatment groups. At the initial pregnancy determination, P/ET differed (P = 0.007) among transfer groups. Concentrations of progesterone tended to be less (P = 0.064) in heifers in the CON group compared to heifers treated with the CIDR (3.6 ± 0.27 compared with 4.4 ± 0.27 ng/mL), and differed between transfer groups (P < 0.001) and days post-estrus (P = 0.019) of the recipients. In summary, while treatment with supplemental progesterone at the time of transfer of IVP embryos using a CIDR increased circulating progesterone, there was no influence on P/ET.

GONADOTROPINA CORIÓNICA EQUINA, ALTERNATIVA DE BAJO COSTO EN TRANSFERENCIA DE EMBRIÓN EN VACAS BAJO PASTOREO TROPICAL

Objetivo: Determinar la eficiencia y costo de dos tratamientos superovulatorios en vacas en pastoreo tropical y sometidas a transferencia de embriones.&#x0D; Diseño/metodología/aproximación: Cincuenta y una vacas distribuidas de forma aleatoria en: Primer grupo, vacas sincronizadas y superovuladas con FSH (200 mg; GFSH, n=9), segundo grupo superovuladas con eCG (2000 UI; GeCG, n=9), tercer grupo (GA. n=16) receptoras de embriones del GFSH y por último cuarto grupo (GB, n=17) receptoras de embriones del GeCG. Se determinó la eficiencia superovulatoria, costo de la transferencia de embriones, costo de gestación, mediante la relación entre respuesta al tratamiento, insumos utilizados y precio de insumos. Los datos obtenidos se compararon mediante prueba Mann-Whitney U o t student para grupos independientes, la tasa de gestación mediante una prueba de Chi cuadrada.&#x0D; Resultados: El costo de la transferencia de embriones es menor en vacas superovuladas con eCG que en vacas con FSH (P=0.000). La cantidad de embriones no difirió entre vacas del GFSH y GeCG (P?0.18). El costo/donadora es 10.8 % menor en GeCG vs. GFSH (P=0.000), el costo/embrión viable fue 35.6 % menor en GeCG (P=0.046) vs. GFSH. La tasa de gestación es similar en ambos grupos (GA vs. GB; P=0.73).&#x0D; Limitaciones/implicaciones: El estudio demuestra que se puede sustituir el uso de FSH por la eCG, con la misma eficiencia a menor costo en vacas bajo condiciones de pastoreo tropical.&#x0D; Hallazgos/conclusiones: Una superovulación con eCG disminuye el costo del programa de transferencia de embriones.

The influence of Multiple Ovulation and Nonsurgical Embryo Transfer on pregnancy rate in Holstein Friesian Heifers

This study was carried out by selecting a total number of 17 Holstein Friesian cows divided into two main groups, the first group (4) cows as donors used to take the embryos from after artificial insemination and the second group (13) heifers cows as recipients for embryos. The same techniques were used on donor and recipient cows for the purpose of hormonal preparation in terms of instance synchronization, preparing recipient cows and superovulation for donor cows. Artificial insemination were done in field, and a nonsurgical flushing procedure embryos applied then graded. Donors reflected a good response to superovulation hormonal treatment. The total number of transferable embryos was 13 from four donors. Evaluation of all embryos was excellent quality. The pregnancy rate results for recipient cows was 61.5%. This result is an acceptable as compared to previous studies due to many factors, the study aim was taking into account all these factors, that can have an influence on these programs to obtain genetically improved animals in a shorter time and lower costs.

Germ Cell Transplantation in Avian Species.

Germcell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germcell transplantation systems have been developed, including sperm, oocyte, or germline stemcell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germcell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.

83 Pregnancy losses after transfer of bovine embryos produced by assisted reproductive technologies

The aim of this study was to evaluate risk factors for pregnancy loss in embryo recipients. We evaluated data from 31,792 embryo transfers (ET), including 859 embryos derived in vivo (IVD), 28,814 produced in vitro (IVP), and 2,119 produced by somatic cell nuclear transfer (SCNT), recorded from 2008 to 2012 on a single commercial farm. Embryos were produced by different in vitro embryo production laboratories, but ET was performed by the same team and within the same herd. Recipients were checked for pregnancy (PR) by ultrasonography 23 days after ET and re-examined on Days 60, 90, and 120 of gestation to identify pregnancy losses (PL). Data were analysed for the main effect of embryo production technique upon PL. For IVD and IVP embryos only, additional risk factors were included in the statistical model, including cryopreservation, type of semen (conventional, sex-sorted, reverse sex-sorted), embryo developmental stage, recipient corpus luteum quality score, use of natural oestrus or synchronization protocol, embryo-recipient synchrony, embryo genetics (taurine v. zebu, dairy v. beef), season (summer v. winter), and fetal sex. Data were analysed using the GLIMMIX procedure of the SAS Software (v 9.3, SAS Institute Inc., Cary, NC, USA). Overall PR was 42.1% (13,395 of 31,792) and PL between days 30 and 60, 60 and 90, and &amp;gt;90 were 8.7, 5.3, and 3.5%, respectively (cumulative PL=17.4%). The SCNT embryos had lower PR compared to IVP and IVD embryos (36.6, 42.5, and 44.2%, respectively; P&amp;lt;0.001) as well as a greater PL in all periods (30-60=45.2, 6.4, and 8.2%; 60-90=3.5, 1.4, and 1.8%; and &amp;gt;90=21.4, 6.3, and 7.1%, respectively; P&amp;lt;0.0001). The PR was similar between fresh and frozen embryos, but lower for vitrified ones (42.9, 43.1, and 39.1%, respectively, P&amp;lt;0.05). Slow freezing resulted in a greater total PL compared to fresh embryos (18.0v. 13.9%, respectively; P&amp;lt;0.05). We observed an interaction between embryo genetics and season. The taurine dairy subgroup had the lowest PR during summer (37.7v. &amp;gt;41.7% in all other groups; P&amp;lt;0.01), but within this group PR increased during winter (37.7v. 44.6%, P&amp;lt;0.05). The PL was greater for taurine than zebu during summer (17.8v. 14.0%; P&amp;lt;0.001), but did not differ during winter (P&amp;gt;0.05). The type of semen, embryo developmental stage, corpus luteum quality score, and embryo recipient synchrony affected pregnancy rates (P&amp;lt;0.05) but not pregnancy losses (P&amp;gt;0.05). Interestingly, the use of ovulation synchronization protocols resulted in similar pregnancy rates but greater pregnancy losses compared to natural oestrus (41.8v. 43.2%, P&amp;gt;0.05; and 15.9v. 22.1%, P&amp;lt;0.05, respectively). Pregnancy loss after 60 days was similar between female and male fetuses (5.0v. 4.9%, respectively; P&amp;gt;0.05). In summary, the assisted technology used to produce and freeze embryos was the main cause of variation in pregnancy losses, which were also affected by the interplay of season by genetic background and by the use of synchronization protocols. Conversely, factors known to affect pregnancy rates, such as embryo developmental stage, corpus luteum quality, and embryo-recipient synchrony, had no effect upon pregnancy loss. Research was supported by Mineirembryo, CAPES, and FAPDF.

110 First ovum pickup-in vitro-produced lidia breed calves using Lidia breed recipients: influence of age and state of the recipients and in vitro-produced embryos on pregnancy rates

We previously demonstrated the success of in vitro embryo production (IVP) in Lidia breed cattle (Gamarra Lazo et al. 2017 Reprod. Fertil. Dev. 30, 187). As in other species, the success of IVP is linked to the birth of calves from this technique. In the Lidia breed, an important factor to consider is the use of Lidia recipients in order to keep the temperament characteristic of this breed to next generations. The aim of the study was to produce ovum pickup (OPU)-IVP calves in the Lidia breed and to assess the effects of recipient and embryo related factors (status of the recipients; development stage of IVF embryos) on pregnancy rate following embryo transfer. Ovum pickup-IVP embryos from Lidia breeds were produced by a standard protocol (Gamarra Lazo et al. 2017 Reprod. Fertil. Dev. 30, 187). Numbers of blastocysts and expanded blastocysts were recorded on Day 7. A total of 27 blastocysts (B) and 34 expanded blastocysts (EB) of excellent quality (grade 1 according to IETS classification) were selected for fresh transfer. All embryos were transferred to Lidia breed recipients (heifers or cows) by a single operator under similar environmental and field conditions. Recipients were synchronized by subcutaneous insertion of an ear implant of 3.3mg of Norgestomet (Crestar®, MSD, Courbevoie, France) for 9 days. Two days before implant withdrawal, 0.5mg of Cloprostenol (Estrumate®, MSD) was injected. No oestrous detection was performed and synchronized females were selected as recipients when they presented a well developed corpus luteum at Day 9 after implant withdrawal (Day 6 to 7 after the expected oestrus). Blood samples were collected from recipients to determine pregnancy status using the bovine pregnancy associated glycoprotein (Idexx, Westbrook, ME, USA) 50-60 days after transfer. Pregnancy rates were analysed by chi-square analysis to compare results between heifers and cows and between B and EB embryo stages. The overall pregnancy rate after transfer of IVP fresh embryos from Lidia breed averaged 41.0% (n=25). A higher pregnancy rate was achieved in cows compared to heifers [51.2% (21/41) v. 20.0% (4/20) respectively, P&amp;lt;0.05]. There was no difference in pregnancy rate between grade 1B [37% (10/27)] and EB [44.1% (15/34)] embryos (P&amp;gt;0.05). Surprisingly, these results suggest that Lidia breed cows are the best recipients for OPU-IVP embryos. This may be related to the limited feasibility of manipulating the uterine horn during the embryo transfer in Lidia breed heifers, which have a low weight (less than 280kg) and present a narrow rectum diameter. It has been also observed that the cervix is very thin and difficult to cross, thus increasing the stress and potentially inflammatory and immune products secretion. Development stage of embryos did not affect pregnancy rate. To our knowledge, no OPU-IVP Lidia breed calves have been reported previously following transfer into Lidia breed recipients. In the current work, 13 OPU-IVP Lidia breed calves were born. Therefore, we confirmed the possibility of applying OPU-IVP and embryo transfer techniques in this breed within a genetic program.

89 Effect of embryo transfer and recipient breed on offspring performance in equine show jumping

Maternal preconceptional, gestational, and postnatal environments alter offspring phenotype in humans and animals. In particular, embryo technologies were shown in horses and other species to affect offspring growth and metabolism. Quantitative genetic calculations in a database containing genealogy and a measured trait (performance) of individuals enable geneticists to estimate the respective contribution of (A) genetic aptitude for the trait (inherited from all parents), (B) genetic maternal aptitude (additional contribution of the mother and her ancestors, acting through pregnancy, lactation, and nursing), and (C) the permanent maternal environment (similarities between the offspring of a dam that are not attributed to genetics) to the variability of this trait in this population. Additionally, fixed effects can be introduced and estimated to take into account additional factors acting on individual performance. The aim of this project was to apply this method to estimate the respective contributions of A, B, and C as well as the fixed effects of maternal parity (D), age at foaling (E), foaling interval (F), embryo transfer (G), and breed of recipient mare (for ET recipients) on the adult offspring sport performance in show jumping horses. The data came from the French Horse Database established in 1976 for genealogy and performance; calculation was done using WOMBAT® software. The performance of 207,984 horses (4.5 years per horse), born to 90,438 dams (of which 4187 were ET recipients, the others being bred by AI or natural service), were studied. Performance was expressed as follows. For each event, points were distributed depending on the horse’s rank within the event and the event’s difficulty. The logarithm of the sum of points of the year has a Gaussian distribution and is then normalised to mean=0 and standard deviation=1. The genetic maternal aptitude and permanent maternal environment showed a small but significant contribution to the variance of performance (1.4 and 1.5%, respectively) compared to a direct genetic contribution of 24.4%. Within each age class of dam, offspring of primiparous mares have a handicap (−0.04 to −0.06) compared to multiparous mares. Within multiparous mares, a foaling interval of 1 year only provided a small advantage (+0.01, nonsignificant) compared to longer intervals (&amp;gt;2 years). The effect of embryo transfer was strong and significant (+0.24), which could reflect the better management of the horses produced by embryo transfer because of their genetic and financial value rather than a physiologic effect. The performance of offspring born to draft and warmblood recipients (+0.27v. +0.24, nonsignificant) were not different. In conclusion, although jumping performance occurs several years after birth, maternal effects are still measurable and embryo transfer is associated with a strongly increased performance independent of genetic value, probably due to better management of these horses. No effect of recipient mare’s breed was found.

Transcriptomics-genomics data integration and expression quantitative trait loci analyses in oocyte donors and embryo recipients for improving invitro production of dairy cattle embryos.

In this paper we first provide a brief review of main results from our previously published studies on genome-wide gene expression (transcriptomics) in donor and recipient cattle used in invitro production (IVP) of embryos and embryo transfer (ET). Then, we present novel results from applying integrative systems genomics and biological analyses where transcriptomics data are combined with genomic data in both donor and recipient cattle to map expression quantitative trait loci (eQTLs). The eQTLs are genetic markers that can regulate or control the expression of genes in the entire genome, via complex molecular mechanisms, and thus can act as a powerful tool for genomic and gene-assisted selection. We identified significant eQTLs potentially controlling the expression of 13 candidate genes for donor cow quality (IVP parameters; e.g. cyclin B1 (CCNB1), outer dense fiber of sperm tails 2 like (ODF2L)) and 19 candidate genes for recipient cows quality (endometrial receptivity; e.g. ER membrane protein complex subunit 9 (EMC9), mannosidase beta (MANBA), peptidase inhibitor 16 (PI16)). Annotation and colocation of detected eQTLs show that some of the eQTLs are in the same genomic regions previously reported as QTLs for reproduction-related traits. However, eQTLs and the candidate genes identified should be further validated in larger populations before implementation as genetic markers or used in genomic selection for improving IVP and ET performance.

Production of donor-derived offspring by allogeneic transplantation of spermatogonia in Chinese rosy bitterling†.

Many bitterling species are facing extinction because of habitat destruction. Since cryopreservation of fish eggs is still not available to date due to their large size and high yolk content, long-term and stable storage of bitterling genetic resources is currently not possible. We recently discovered that cryopreservation of early-stage germ cells is possible in several fish species and that functional gametes derived from the frozen materials can be produced through their transplantation to embryonic recipients. However, bitterlings have uniquely shaped eggs and their embryos are extremely fragile, making it difficult to perform germ cell transplantation. Therefore, as a first step, we conducted intra-species spermatogonial transplantation using recessive albino Chinese rosy bitterling as donors and wild-type Chinese rosy bitterling as recipients to develop a system to convert freezable early-stage germ cells into functional gametes, particularly eggs. Approximately 3000 testicular cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less recipient embryos produced by dead end (dnd)-knockdown. At 6 months, ten male recipients and nine female recipients produced gametes. Mating studies with the opposite sex of recessive albino control fish revealed that six males and three females produced only albino offspring, suggesting that these recipients' endogenous germ cells were completely removed by dnd-knockdown and they produced only donor-derived gametes. Thus, we successfully established a germ cell transplantation system in an iconic endangered teleost, bitterling. The technology established in this study can be directly applied to produce functional gametes of endangered bitterlings using cryopreserved donor cells.

Factors affecting the pregnancy rate of embryo recipient IVP: literature review

In the last decades, Brazilian livestock farming has undergone great expansion, based not only on quantitative growth, but also on quality. The qualitative growth is based on factors derived from the use of reproductive biotechniques, such as the In Vitro Embryo Production (IVEP), which allows significant genetic advancement in the herd in a reduced period of time, by the multiplication of the genetics of higher animals. For implantation of IVEP, with efficiency and viable cost, it is necessary to consider some factors, as well as the interaction with the gestation rate. The objective of this review was to report the main factors involved in the gestation rate of embryo receptors produced in vitro (IVP), such as corpus luteum quality, donor and recipient synchrony, donor breed, embryo development and quality, reproductive and semen effects, thermal stress, period of the year and number of previous ETs in the recipient. Although the IVEP is an important tool for the genetic improvement of the bovine herd, it is necessary to consider the variations in the results of gestation after its use, mainly with regard to donors and recipients, geographic region, environmental conditions, sanitary and nutritional management of animals, as well as the conditions inherent in the IVP laboratory. The observation of these factors, as well as the constant search for improvements, are fundamental conditions for the success, viability and economic return of IVEP. es fundamentais para o sucesso, viabilidade e retorno economico da PIVE.

NANOG Is Required for the Long-Term Establishment of Avian Somatic Reprogrammed Cells.

SummarySomatic reprogramming, which was first identified in rodents, remains poorly described in non-mammalian species. Here, we generated avian reprogrammed cells by reprogramming of chicken and duck primary embryonic fibroblasts. The efficient generation of long-term proliferating cells depends on the method of delivery of reprogramming factors and the addition of NANOG and LIN28 to the canonical OCT4, SOX2, KLF4, and c-MYC gene combination. The reprogrammed cells were positive for several key pluripotency-associated markers including alkaline phosphatase activity, telomerase activity, SSEA1 expression, and specific cell cycle and epigenetic markers. Upregulated endogenous pluripotency-associated genes included POU5F3 (POUV) and KLF4, whereas cells failed to upregulate NANOG and LIN28A. However, cells showed a tumorigenic propensity when injected into recipient embryos. In conclusion, although the somatic reprogramming process is active in avian primary cells, it needs to be optimized to obtain fully reprogrammed cells with similar properties to those of chicken embryonic stem cells.

Embryo donation and receipt in Australia: views on the meanings of embryos and kinship relations

Research on embryo donation and receipt continues to grow, highlighting how specific national contexts shape views and experiences. The present article reports on a qualitative study on embryo donation and receipt in Australia. Interviews were conducted with 15 participants: embryo donors and those seeking to donate (6), embryo recipients and those seeking donors (3), people with embryos in storage or previously in storage (5), and egg donors where resulting embryos were donated to a third party (1). A deductive thematic analysis identified four key themes: understandings of embryos as cells, potential children, and/or children; a focus on relationships between “siblings”; importance of language and “family words” in discussing relationships; and extended family members having difficulty understanding the concept of embryo donation. The article concludes with a consideration of the implications of the findings in terms of the practice of embryo donation and the policies that surround it.

Short fish-origin DNA elements served as flanking sequences in a knockdown cloning vector enabling the generation of a functional siRNA molecule in mammalian cells and fish embryos

Improving the quality of a siRNA-knockdown cloning vector requires simpler, shorter, and more effective flanking sequences. In this study, we designed such flanking sequences based on those found in zebrafish pre-miR3906, namely, internal element (IE) 1 and IE2. We engineered a vegf-shRNA fragment flanked by an 80-bp IE1/IE2 and then inserted into the 3′ UTR of GFP reporter cDNA driven by a cytomegalovirus promoter to obtain a plasmid containing gfp-IE-vegf-shRNA-polA. Upon microinjection of this plasmid into zebrafish embryos, we found that IE flanking sequences could effectively induce the production of vegf-shRNA fragment, which was then processed into a functional siRNA to silence the target vegf121 gene. Northern blot showed that the vegf-shRNA fragment was cleaved from gfp-IE-vegf-shRNA-polA, resulting in the loss of polyA tails, subsequently degrading the remaining RNA-containing GFP. Moreover, Western blot revealed that addition of IE-based vegf-shRNA fragment could markedly decrease the expression of VEGF. Finally, to facilitate a more versatile application of the IE-based knockdown vector, we generated an inducible expression vector in which IE-vegf-shRNA was constructed downstream in a Tet-on system to generate a Tet-on-IE-vegf-shRNA construct. After doxycycline induction, the protein level of VEGF in SW620 cells harboring the Tet-on-IE-vegf-shRNA construct was decreased 77%. Interestingly, when SW620 cells harboring Tet-on-IE-vegf-shRNA cells were induced and transplanted into zebrafish embryos, we found that abnormal branch of the sub-intestinal vessels was reduced in the recipient embryos, suggesting that vegf-shRNA cleaved from Tet-on-IE-vegf-shRNA-polA was processed into a functional vegf-siRNA in embryos suppressing endogenous VEGF and reducing tumor angiogenesis. Therefore, we conclude that fish-origin IEs are flanking sequences with short, simple, and effective DNA elements. This IE-based knockdown cloning vector provides a new alternative material to facilitate the generation of functional siRNA with which to perform loss-of-function experiments, both in vitro (mammalian cells) and in vivo (zebrafish embryos).

Receiving, or ‘Adopting’, Donated Embryos to Have Children: Parents Narrate and Draw Kinship Boundaries

Existing research suggests that embryo donation (ED) may be seen as similar to adoption by those who donate or receive embryos, or it may not. Our qualitative study explored whether having a child via embryo donation initiated kinship connections between embryo donor and recipient families as interpreted by recipient parents. Interviews were conducted with five parents from four families whose child(ren) had been born via embryo donation. All four families had an open-contact relationship set up with the couples who donated their embryos through an agency in the USA. Narrative thematic analysis of interview data and visual family map drawings were used to explore kinship conceptualizations. We conclude that the dilemma experienced by parents who have a child via embryo donation is to decide how to reconcile their child’s different genetic heritage, when gestation and upbringing both clearly boundary family membership solely within the recipient family. While some families were still struggling with this dilemma, one solution embarked upon by some parents when drawing their family map was to expand family membership, not only on the basis of genetics, but also via an appreciation of shared family and community values too.