Non-surgical Embryo Transfer Research Articles

What has embryo recovery and transfer taught us about equine reproductive physiology?

Equine embryo transfer has been the foundation stone on which modern reproductive technologies and novel research in this species has been built. Nonsurgical embryo recovery and transfer methods developed from the original surgical methods, and the ease with which these procedures can be undertaken has enabled these techniques to play a continuing role in original research. We document how embryo recovery and/or transfer have increased our knowledge about many of the events involved in equine reproductive physiology. These include, when the equine embryo enters the uterus, the importance and development of the blastocyst capsule, secretory products of early embryos, early maternal-embryo interactions, effects of uterine asynchrony on early embryo development, the role of endometrial cups and maternal immunological tolerance of extraspecies embryo transfers, and the influence of uterine environmental on fetal growth and development.

Is the corpus uteri a suitable site for embryo transfer in the pig?

Numerous attempts to establish a practicable means of non-surgical embryo transfer in pigs were unsuccessful until instruments were developed to permit deposition of embryos deep inside a uterine horn. Recently, however, successful non-surgical transfer of embryos to the uterine body in non-sedated recipients has been reported. These reports have prompted us to present data from a study that addresses the question of whether the commonly experienced low pregnancy rates following transfers to the corpus uteri results from the inability of endometrial receptors to recognize the presence of embryos or to the prevailing unfavorable local conditions. Blastocysts were transferred to the corpus uteri of recipients in which maintenance of pregnancy was assured by the transfer of additional embryos to the tip of a uterine horn. Embryos from pigmented and unpigmented donors were used to be able to differentiate between fetuses from embryos transferred to different sites. Fewer fetuses developed from embryos transferred to the corpus uteri than from embryos transferred to the uterine horn. This leads to the conclusion that prolonged retention of blastcysts in the uterine body under conditions inappropriate for their stage of development is responsible for an unsatisfactory pregnancy rate.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

72 Seminal Plasma Uterine Priming Reduces Offspring Growth and Alters Uterine Artery Indices of Vascular Resistance in Embryo-Recipient Beef Cows

Abstract Uterine priming with seminal plasma has been shown to alter endometrial gene expression as well as uterine cytokines and chemokines. However, the effects of seminal plasma uterine priming on uterine blood flow, fetal and postnatal offspring growth performance have yet to be elucidated. Therefore, we investigated the effects of pooled seminal plasma uterine priming at estrus on embryo crown-rump length, uterine blood flow, and birth weights. Commercial cows (n = 65) were synchronized, evaluated for standing estrus (day 0), and randomly assigned to treatment groups: seminal plasma or control. Seminal plasma treated cows (n = 27) received 0.5 mL of pooled seminal plasma from commercial bulls mismatched from embryo sire placed in their uterine body via artificial insemination rod2 hours after estrus detection (day 0). Control cows (n = 27) were passed through the chute without receiving treatment. On day 7, cows underwent non-surgical embryo transfer and were confirmed pregnant on day 35 via ultrasonography. Final treatment numbers were n = 9 seminal plasma and n = 7 control. On days 35, 40, and 45 embryo crown-rump length was measured via transrectal ultrasonography. On days 140, 180, 200 and 220, uterine artery hemodynamics were measured via Doppler ultrasonography. Birth weights were collected within 24 hours of birth. Data collected over time were analyzed using repeated measures of ANOVA with fixed effects of treatment, day, and their respective interaction. Birth weights and gestation length were analyzed using ANOVA. Embryo sire and dam, side of pregnancy, and fetal sex were included as covariates if P < 0.10. Covariance structure was selected based on lowest AIC and BIC. Embryo crown-rump length was decreased (P = 0.0483) among seminal plasma treated cows compared with controls. Embryo crown-rump length increased (P < 0.0001) by day. Total uterine blood flow increased (P < 0.0001) as gestation proceeded. Ipsilateral resistance index was increased (P = 0.0431) among seminal plasma treated cows compared with controls. Ipsilateral resistance index was increased (P = 0.0107) on day 180 of gestation compared with days 200 and 220 of gestation. Ipsilateral pulsatility index was decreased (P < 0.0001) on day 220 of gestation compared with days 140 and 180 of gestation. Contralateral blood flow was decreased (P = 0.0037) on day 140 of gestation compared with days 180, 200, and 220 of gestation. There were no differences in contralateral uterine artery blood flow, resistance index, or pulsatility index, or gestation length between treatment groups. Birth weights were decreased (P = 0.0102) among calves from seminal plasma treated cows compared with calves from control cows. In summary, seminal plasma uterine priming reduced offspring growth while increasing uterine artery resistance index, which indicates uterine vascular bed anomalies that persisted into the third trimester of pregnancy.

- Invited Review - Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm.

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

Hormonal-Induced Cervical Relaxation During Diestrus in Ewes: Cervical Transposing Feasibility and Use of hCG for Rescuing Disrupted Luteal Function

ABSTRACT Cervical relaxation (CR) was performed in ewes during diestrus, to prospect a feasible protocol for non-surgical embryo transfer (NSET). In Trial 1, naturally mated ewes (n=13) received CR protocols with estradiol benzoate (EB, 1 mg on D6) and oxytocin (OT, 50 IU on D7) only (G-EB+OT) or associated with human chorionic gonadotropin (hCG, 300 IU on D7, G-EB+OT+hCG) and were compared to non-hormonally treated (G-control) ewes. Estradiol concentration increased (P<0.05) from D6 to D7 in G-EB+OT and G-EB+OT+hCG, then decreased (P<0.05) on D8. The G-EB+OT had reduced (P<0.05) progesterone on D8, and 0% (0/5) pregnancy, while G-control had 100% (3/3) conception rate. The treatment with hCG mitigated the progesterone drop on D8 and resulted in 50% (2/4) conception rate. In Trial 2, cervical transposing success after treatment with EB, OT, and hCG on D6 and D7 was assessed in nulliparous (n=25) and multiparous (n=30) ewes. The EB+OT+hCG protocol allowed efficient cervical transposition (~78% of cervical transposition success into ~5 min) in nulliparous and multiparous females and did not alter total and blood perfusion areas of corpora lutea (CL). The use of EB+OT+hCG was efficient to ensure effective cervical transposing in nulliparous and multiparous ewes, with no apparent deleterious effect on CL function, and may contribute to animal welfare, allowing a less invasive NSET procedure.

Genomic selection in beef cattle creates additional opportunities for embryo technologies to meet industry needs.

The use of genotype information to improve the predictability of Expected Progeny Difference was first implemented in American Angus cattle in 2009 and has now grown to where over 50% of all registered calves are genotyped. Animals with only a genotype now have genetic prediction accuracy equivalent to eight or more progeny records across all traits. Reproductive technologies have also been widely adopted with approximately 50% of all calves born being the result of artificial insemination. Non-surgical embryo transfer started increasing in the mid 1990s with just over 10% of calves born being the result of embryo transfer since 2005. The number of embryos created with in vitro technologies has risen sharply since 2015 and now accounts for close to 30% of all ET calves. Genomics has enabled embryo technologies to be more impactful, as females can be selected with greater accuracy and sires can be used at earlier ages with moderate accuracy. Large numbers of females genotyped each year also increases the number of selection candidates, increasing the selection intensity. Genomics, combined with increased recording, also provides more information on females. This increases the spread in the estimated index values of current dams, identifying more elite dams for selection as embryo donors. The greater scope of female selection also contributes to better inbreeding management. Commercial animals genotyped could be targeted for oocyte harvesting at slaughter, creating opportunities for low cost high value beef embryos to be used in the beef on dairy segment of the industry.

Assisted reproductive techniques for canines: preservation of genetic material in domestic dogs.

Assisted reproductive techniques (ARTs), such as artificial insemination, in vitro fertilization, and cryopreservation of gametes/zygotes, have been developed to improve breeding and reproduction of livestock, and for the treatment of human infertility. Their widespread use has contributed to improvements in human health and welfare. However, in dogs, only artificial insemination using frozen semen is readily available as an ART to improve breeding and control genetic diversity. A recent priority in sperm cryopreservation is the development of alternatives to egg yolk, which is widely used as a component of the sperm extender. Egg yolk can vary in composition among batches and is prone to contamination by animal pathogens. The latter can be a problem for international exchange of cryopreserved semen. Low-density lipoprotein and skim milk are promising candidates for use as extenders, to ensure fertility after artificial insemination. Although not tested for its effects on fertility following artificial insemination, polyvinyl alcohol may also be a useful alternative to egg yolk as an extender. The development of cryopreservation techniques for canine embryos lags behind that for other mammals, including humans. However, given the success of non-surgical embryo transfer in 2011, studies have sought to refine this approach for practical use. Research on sperm cryopreservation has yielded satisfactory results. However, investigation of other approaches, such as cryopreservation of oocytes and gonadal tissues, remains insufficient. Techniques for the efficient induction of estrus may aid in the development of successful canine ARTs.

Improved Non-Surgical Embryo Transfer Method using Porcine Vitrified Blastocysts

Improved Non-Surgical Embryo Transfer Method using Porcine Vitrified Blastocysts

A Nonsurgical Embryo Transfer Technique for Fresh and Cultured Blastocysts in Rats.

The use of a nonsurgical embryo transfer technique in rodents eliminates the potential pain, distress, and health complications that may result from a surgical procedure and as such, represents a refinement in rodent assisted reproductive techniques. A nonsurgical technique has not been previously developed for use with rat embryos. Here we describe an efficient method to deliver either fresh or cultured blastocyst stage embryos to the uterine horn of pseudopregnant female rats using a rat nonsurgical embryo transfer (rNSET) device. The rNSET device is composed of a Teflon catheter and a hub that attaches to a 2 μL pipette. Oxytocin is used to dilate the cervix before the delivery of blastocysts, allowing passage of the rNSET catheter directly into the uterine horn for embryo delivery. The efficiency of recovery of pups after nonsurgical embryo transfer is similar to the efficiency after surgical embryo transfer. Furthermore, the technique is not stressful to the subjects, as demonstrated by the absence of a decrease in weight or increase in fecal corticosterone level in recipients of embryos delivered nonsurgically, without the use of anesthesia or analgesia.

Factors affecting pregnancy rates for goat embryos recovered and transferred by transcervical route

Abstract This study evaluated the factors affecting non-surgical embryo transfer (NSET) efficiency in goats. Donor goats (n = 10) were superovulated, mated and embryos were collected by non-surgical embryo recovery 7 d after estrous onset. A total of 28 pluriparous recipient goats were subjected to short-term progestagen-based estrous induction protocol with equine chorionic gonadotropin. They received 36 fresh embryos as single (n = 20) or in pairs (n = 8) by NSET 6 to 7 d after estrous onset, ipsilateral to the corpora lutea (CL). Pregnancy rate was affected (P 0.05) by the uterine horn, number of CL (1 to 3), lactational status and number of embryos transferred per recipient goats. Both overall pregnancy and kidding rates were 32.1% and 13 kids were born, representing an embryo survival rate of 36.1%. Goat embryo transfer programs can be successfully performed by using non-surgical techniques, achieving satisfactory embryonic survival rates.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

Blastocyst hatching site is regularly distributed and does not influence foetal development in mice

Hatching out from the zona pellucida (ZP) is a crucial step for blastocyst implantation and development. However, it is still unknown whether the location of the hatching site relative to the inner cell mass (ICM) affects embryo implantation and foetal development. Here, we classified hatching blastocysts into three categories, 0° ≤ θ ≤ 30°, 30° < θ ≤ 60°, and 60° < θ ≤ 90°, in which θ is determined based on the relative position of the hatching site to the arc midpoint of the ICM. Non-surgical embryo transfer (NSET) devices were employed to evaluate blastocyst implantation and embryo development. Of 1,827 hatching blastocysts, 43.84%, 30.60%, and 21.67% were categorized as 30° < θ ≤ 60°, 0° ≤ θ ≤ 30°, and 60° < θ ≤ 90°, respectively. Embryos with different hatching sites showed no distinct differences in blastocyst implantation; surrogate female pregnancy; embryo development to term; litter size, or offspring survival, gender, or body weight. Our results indicate that mouse blastocyst hatching site is not randomly distributed. Embryo implantation and development are not correlated with the blastocyst hatching site in mice. Thus, assessment of the blastocyst hatching site should not be recommended to evaluate mouse blastocyst implantation and developmental potential.

Matrix metalloproteinases improves trophoblast invasion and pregnancy potential in mice

Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson’s trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.

Non-surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus.

This study aimed to compare the efficiency of non‐surgical embryo transfer (ET) using a newly developed catheter, which enables transferring embryos into a proximal site of the uterus (mostly uterine body), and surgical ET of vitrified porcine embryos. In Experiment 1, the catheter was inserted into 12 gilts, with each half of the group allocated to skilled or novice operators. The time required for insertion into the uterus did not differ between skilled and novice operators (4 min 9 s and 4 min 6 s, respectively). In Experiment 2, 12 gilts were used as recipients for non‐surgical and surgical ET with vitrified embryos (n = 6, each). There was no significant difference in the rate of piglet production based on the number of transferred embryos between surgical and non‐surgical ET (25.8% vs. 15.4%, p = .098). The results suggest that non‐surgical ET catheter allowed for easy insertion and transfer of embryos without special training. Although the catheter is effective for deposition of embryos into the proximal site of uterus, the efficiency of piglet production is not enhanced compared with surgical ET. The ET method using this catheter, being labor‐saving and less‐invasive, may contribute to the improvement of ET in pigs.

Piglet production by non-surgical transfer of vitrified embryos, transported to commercial swine farms and warmed on site.

We investigated the feasibility of piglet production by non‐surgical embryo transfer (Ns‐ET) of vitrified porcine blastocysts and expanded blastocysts transported to commercial farms and warmed on site (V/T/W embryos). Ns‐ET was performed by depositing 11–20 vitrified and warmed embryos at a proximal site within the uterus via a catheter. In Experiment 1, the effect of donor–recipient estrous cycle asynchrony on the efficiency of Ns‐ET of vitrified and ordinary warmed embryos was investigated at the experimental facility. With a 1‐day delay recipients relative to that of donor, the farrowing rate was 50.0% and the survival rate to term was 21.1%. In Experiment 2, Ns‐ET using recipients with a 1‐day delay and vitrified embryos after one‐step warming and dilution was evaluated at the experimental facility. Although the resulting farrowing rate was 42.9%, the survival rate was 6.4%. In Experiment 3, Ns‐ET was conducted using V/T/W embryos at four commercial farms, where piglets derived from them were produced. When artificial insemination was conducted prior to Ns‐ET, the farrowing and survival rates obtained using V/T/W embryos were 75.0%, and 21.3%, respectively. These results show that Ns‐ET of V/T/W embryos using this protocol would be feasible for piglet production at farms.

Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically.

This study was performed to evaluate reproductive performance after non‐surgical embryo transfer (Ns‐ET) of 10–15 porcine expanded blastocysts (ExBs) that had been vitrified and warmed (V/W) using the micro volume air cooling (MVAC) method. The effect of asynchrony between the donor and recipient estrous cycle was investigated. Ns‐ET was conducted in recipients whose estrous cycle was asynchronous to that of donors by a delay of 2, 1, or 0 days. In the 2‐day and 1‐day groups, the similar farrowing rates (27.3% and 25.0%) and survival rates to term (13.9% and 15.7%) were obtained after Ns‐ET of V/W ExBs. None of the recipients in 0‐day group farrowed. Artificial insemination (AI) prior to Ns‐ET was then evaluated. Ten–15 V/W ExBs were transferred non‐surgically to 12 recipients whose estrous cycles were asynchronous to that of donors by a 2‐day delay. All of the recipients produced piglets, and all (100.0%) delivered piglets were derived from the transferred V/W ExBs. The survival rate of V/W ExBs to term was 25.2%. These results demonstrate that Ns‐ET of V/W ExBs using MVAC can facilitate piglet production, even if 10–15 embryos are transferred. Moreover, piglets were obtained stably when AI was performed prior to Ns‐ET.

Goat incubator: Can bovine oocytes be matured in the uterine horn of a goat?

We used a goat as a live incubator, along with associated nonsurgical embryo transfer techniques, to perform ex situ (in vivo) maturation of bovine oocytes. Immature bovine cumulus-oocyte complexes (COCs) aspirated from 3-8 mm follicles from slaughterhouse ovaries were randomly split into two groups for in vitro (IVM; n = 38) and ex situ maturation (ESM; n = 40). IVM was performed for a period of 24 h at 38.5 ºC and with 5% CO2 in the air of maximum humidity. For ESM, a presynchronized nulliparous goat (12 months old) received 40 immature COCs in the uterine horn apiece, via the transcervical route. After 24 h the structures were retrieved through uterine flushing. Analyses of nuclear maturation and lipid quantification were performed on oocytes from both groups. Fluorescent intensity was compared using the Student’s t-test. Forty-seven percent of the structures were recovered after uterine flushing (19/40). The nuclear maturation rate was 94.5% (18/19) and 81.6% (31/38) for the ESM and IVM groups, respectively. In vitro-matured COCs contained more lipid droplets, expressed as a higher amount (p &lt; 0.05) of emitted fluorescent light than ex situ-matured COCs (858 ± 73 vs. 550 ± 64 arbitrary fluorescence units, respectively). This is the first report to associate nonsurgical embryo transfer techniques and a goat as a live incubator for the maturation of bovine oocytes. We conclude that bovine oocytes can progress meiotically in the uterus horn of a goat and that transcervical transfer of bovine oocytes to a goat’s uterus could present an alternative to nuclear maturation.

Factors influencing the commercialisation of cloning in the pork industry

Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.

Cryopreservation and transfer of pig embryos

Recent advances in cryopreservation and non-surgical transfer of pig embryos have made embryo transfer in pigs a commercially viable technology especially for the international transfer of valuable genetic material. Early research demonstrated that early stage pig embryos were highly sensitive to temperatures below 15°C and this sensitivity decreased with development to peri-hatching blastocysts which is accompanied by a reduction in lipid content. Removal of the lipid prior to freezing was found to improve the freezability and resulted in the birth of piglets. It was shown that by polarising the lipid material out of the blastomeres and using ultra rapid freezing (vitrification) the early stage zona pellucida intact embryo would also survive freezing and result in the birth of piglets. Other factors that have contributed to successful cryopreservation of the early stage pig embryo include the use of cytoskeletal stabilising agents, specific cryoprotectants, increasing cooling rates using open pulled straws or micro droplets and assisted hatching. The present review discusses the relative importance of each of these factors. The development of non-surgical transfer technology and its importance in the application of embryo transfer in the pig industry is also reviewed.