Non-surgical Embryo Transfer Research Articles

39 CHROMOSOME ABNORMALITIES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY 'HANDMADE CLONING'

Embryos produced by nuclear transfer using somatic cells as donors exhibit a lower developmental competence compared to in vivo developed and IVM/IVF/IVC embryos. The so-called handmade somatic cell nuclear transfer (HSCNT or handmade cloning) that was recently presented (Vajta et al. 2003 Biol. Reprod. 68, 571–578) has, however, improved the reconstructed embryo and subsequent blastocyst rate. Further, the high total cell number (n = 216) and the high ratio of cells allocated to the inner cell mass (35%) as well as the initial pregnancy rate of 48% on Day 28 following nonsurgical transfer of HSCNT embryos indicated a high average quality. But still the pregnancy loss of HSCNT embryos was high and resulted in a birth rate of 8%. The aim of this study was to estimate the chromosomal variation in HSCNT embryos using fluorescent in situ hybridization (FISH) and to evaluate this as an additional parameter of embryo quality. Nuclei from 49 Day 7 HSCNT embryos from five independent trials were isolated by hypotonic treatment and fixed. Then the nuclei were hybridized with differentially labelled cJAB8 and p33E39 probes that hybridize specifically to the centromeric region of chromosome 6 and 7 (Viuff et al. 2000 Biol. Reprod. 63, 1143–1148). A total of 6715 nuclei were analyzed for chromosomal abnormalities, and the percentage of nuclei with false negative scores represented 1.8%. Only 4.1% of the embryos (2 of 49) had a completely normal diploid composition, while the remaining 47 embryos exhibited different types of mixoploidy. Of the 47 mixoploid embryos, 87% contained more than one type of chromosomal variation with diploid/triploid/tetraploid being the most frequent constitution (43% of the 47 mixoploid embryos). No pure polyploid embryos were observed. The percentages of HSCNT embryos in the groups of 0%, 1–25%, and 26–100% polyploid cells were 4.1%, 67.5%, and 28.6%, respectively. This is significantly higher than the corresponding figures produced and analyzed by comparable methodology of bovine IVM/IVF/IVC embryos (Viuff et al. 1999 Biol. Reprod. 60, 1273–1278). However, 71.4% of the HSCNT embryos contained less than 25% polyploid cells; this low level may not have compromised the developmental competence of these embryos.

Perspectives for artificial insemination and genomics to improve global swine populations

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future.

An improved method for nonsurgical embryo transfer in the mare

Equine Veterinary EducationVolume 16, Issue 1 p. 39-44 An improved method for nonsurgical embryo transfer in the mare S. Wilsher, S. Wilsher University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UKSearch for more papers by this authorW. R. Allen, Corresponding Author W. R. Allen University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UK*University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UKSearch for more papers by this author S. Wilsher, S. Wilsher University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UKSearch for more papers by this authorW. R. Allen, Corresponding Author W. R. Allen University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UK*University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UKSearch for more papers by this author First published: 05 January 2010 https://doi.org/10.1111/j.2042-3292.2004.tb00265.xCitations: 28AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume16, Issue1February 2004Pages 39-44 RelatedInformation

Reproduction results and offspring performance after non-surgical embryo transfer in pigs

Increased interest in transfer of valuable genetic material around the world with minimal health risks has stimulated the development of non-surgical embryo transfer (nsET) technologies in pigs. Experimental evidence shows that nsET without sedation of the recipients is now feasible. The goal of this study, therefore, was to evaluate a method of nsET under commercial conditions. The experiment included 135 donor gilts and 45 multiparous recipient sows. Ovulation was induced in both donors and recipients, and nsET was performed using the Swinlet® catheter. Donor gilts averaged 16.5 (7–45) corpora lutea, but this depended on age of the donor (P<0.05). An average of 10.1 transferable blastocysts was recovered per donor, and the recovery rate was 84%. For 44 nsET, 14 recipients (31%) came into estrous before Day 23 after ovulation, 7 recipients (16%) came into estrous between Days 23 and 30, 3 recipients (6.8%) came into estrous between Days 39 and 48, 2 recipients (4.5%) had a late abortion. Finally, 18 of 44 recipients (41%) resulted in successful births, with an average liter size of 7.2±2.8. Birth weight of nsET piglets were 0.2kg more than control piglets, but depended on litter size (P<0.05). The sex-ratio was not different from 50%. No anatomical abnormalities were observed in the offspring of nsET. Of the recipients that did not become pregnant from nsET, 91% became pregnant after insemination in the next estrous. Gilts born from nsET gave on average 12.4±3.0 total born piglets in their first pregnancy. In conclusion, the nsET procedure used in this study can be applied in practice without the need for special facilities, such as surgical and anesthesia equipment.

285BIRTH OF PIGLETS AFTER NON-SURGICAL TRANSFER OF PORCINE EMBRYOS CULTURED IN PZM-4 WITH ALTERED CONCENTRATIONS OF AMINO ACIDS

We previously developed an in vitro production (IVP) system for porcine embryos and obtained piglets after surgical transfer of blastocysts cultured in Porcine Zygote Medium (PZM)-4. However, the developmental competence of pig IVP embryos to the blastocyst stage is still low and further improvement of IVC medium is needed. In the present study, we evaluated the effects of the addition of glutamine (Gln), hypotaurine (HT), taurine (Tau), BME-essential (EA) and MEM-nonessential (NA) amino acids solutions to PZM-4, and the replacement of polyvinyl alcohol (PVA) with BSA on embryo development to blastocysts. Moreover, the developmental competence of IVP blastocysts after nonsurgical embryo transfer (NS-ET), using a flexible catheter (FC) for deep intrauterine insemination, was investigated. Porcine COC from prepubertal gilts were matured and fertilized in vitro, using frozen-thawed ejaculated boar semen. Presumptive zygotes were cultured in PZM-4, as a basal culture medium, until Day 5 after IVF. Data from six replicates were analyzed by ANOVA. Addition of 0.25 to 4mM Gln to PZM-4 (containing 5mM HT) significantly increased the percentage of embryos that developed to blastocysts (15 to 31%), with addition of 2mM Gln significantly increasing the total cell numbers in blastocysts (43±17 cells) compared with no addition (3% and 20±4 cells, respectively). Addition of 1.25 to 10mM HT to HT-free PZM-4 supplemented with 2mM Gln (named PZM-5) significantly increased the percentage of embryos that developed to blastocysts (22 to 28%) compared with control (no HT;; 4%). In the culture with HT-free PZM-5, addition of 5mM Tau significantly increased blastocyst yield (17%) compared with control (4%). However, Tau addition in the presence of 5mM HT had no effect on development to the blastocyst stage. In combinations of EA and NA added to PZM-5, a single dose of EA significantly increased the percentage of embryos that developed to blastocysts (27%) compared with no dose (19%) or with a double dose of EA (20%), while a double dose of NA significantly increased the total cell numbers in blastocysts (43±16 cells) compared with no NA (37± 6 cells). Replacement of PVA with BSA in PZM-5 had no effect on embryo development to the blastocyst stage. Crossbred sows were used as recipients for NS-ET, and had their estrous cycle synchronized by a described previously method (Yoshioka et al., 2002 Biol. Reprod. 66, 112–119). Five days after hCG injection, a FC was introduced via the cervix into the uterine horn of recipients without sedation. Day-5 blastocysts cultured in PZM-5 were then transferred together with 5ml of TALP-Hepes (45 to 50 blastocysts/recipient). Of 6 recipients, one sow became pregnant and farrowed 7 piglets. Our results indicate that the addition of amino acids to PZM-4 can improve porcine embryo development to the blastocyst stage, and that blastocysts cultured in a chemically defined medium, PZM-5, can develop to full-term following NS-ET.

Mammalian embryo culture in a microfluidic device.

Over the last decade, the use of in vitro production of mammalian embryos and the utilization of assisted reproductive technologies (ART), such as non-surgical embryo transfer, cryopreservation, and intracytoplasmic sperm injection (ICSI), has increased. However, the efficiencies of ART remain low. Currently, millions of couples in the United States are affected by infertility and seek treatment. More than 16,000 babies in the United States, each year are born using ART such as in vitro fertilization (IVF), assisted hatching, and ICSI (). A critical area for ART is culturing embryos and evaluating their morphology. There have been some advances made in embryo culture medium; however, the efficiency of embryo culture remains low. Additionally, embryo-handling techniques and tools for culture and evaluation have not changed for several years. The growth of miniaturization technologies toward miniature mechanical and fluidic systems has created opportunities for fresh examination of basic embryo physiology. There are many existing limitations in mammalian embryo-handling procedures that may be addressed with these new miniaturization technologies.

295POSSIBLE DOPAMINERGIC MECHANISM REGULATING THE EQUINE CORPUS LUTEUM

Timing of embryo transfer to recipient mares is crucial to the success rate of an embryo transfer (ET) program. Recipient mares need a functional corpus luteum (CL) to maintain the early stages of pregnancy. Interference with luteal function appears to be a significant cause of failure with nonsurgical ET. Little is known about the endocrine control of luteal function in the mare, and the possibility of neuronal control of equine luteal function has not yet been studied. Dopamine (DA) has been shown to affect progesterone secretion in the bovine CL. Prior research in our laboratory suggested the possibility of dopaminergic regulation in the equine CL. The aim of this study was to document the presence of DA D1 receptor (D1r) and DA D2 receptor (D2r) within the equine CL. Immunocytochemistry (ICC) was performed on sections from 9 corpora lutea collected from a local equine abattoir. Tissues were stained using the avidin-biotin complex (ABC) method (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Tissue sections were deparaffinized and rehydrated;; endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide in methanol at room temperature. To decrease nonspecific staining, tissue sections were incubated in goat serum (Vectastain ABC Elite kit). Tissue sections were then incubated overnight at 4°C with primary antibodies: Rabbit anti-Dopamine D1 receptor (Calbiochem, San Diego, CA, USA) or Rabbit anti-D2 receptor polyclonal antibody (Chemicon International, Inc, Temecula, CA, USA). Sections were then washed in PBS and incubated in biotinylated goat anti-rabbit IgG secondary antibody. After washing in PBS, sections were incubated in ABC. Sections were washed in PBS and the signal was visualized using 3-amino-9-ethylcarbazole (AEC Red, Vector Laboratories, Burlingame, CA, USA). Tissues were then counterstained with Immunomaster Hematoxylin (American Master Tech Scientific, Inc., Lodi, CA) to visualize the nuclei. As a negative control, tissues were incubated in normal rabbit serum instead of primary antibody, and as a positive control, the ICC procedure was performed on whole rat brain slices. Significant staining of luteal cells was observed using the D1r and D2r antibodies. Positive staining for D1r and D2r was seen throughout luteal cells;; however, no nuclear staining was observed. The presence of these receptors in equine CL tissue suggests a functional significance for DA in luteal function. Further research needs to be performed to determine the mechanistic function of dopamine in mare reproduction.

Successful nonsurgical deep uterine embryo transfer in pigs

At present, it is possible to transfer pig embryos directly into the uterine body of sows by nonsurgical procedures. The aim of this study was to develop a procedure for nonsurgical embryo transfer (ET) into the upper part of one uterine horn in gilts and sows. In experiment 1, 29 gilts and 43 sows were used. Intrauterine insertions took place for each female at days 4–6 of the estrous cycle (D0=onset of estrus). An artificial insemination (AI) spirette was inserted into the cervix to assist with the guidance of a modified flexible catheter originally developed for deep intrauterine insemination in pigs. The flexible catheter length inserted anterior to the inserted AI spirette was 43.0±1.7 cm. The time required to complete the procedure was affected by the type of female ( P<0.001) and by the difficulties encountered for inserting the catheter ( P<0.001). However, when no or minor difficulties were encountered during the insertion of the catheter (in approximately 70 and 80% of gilts and sows, respectively), the time required to complete the procedure did not differ between gilts (2.5±0.1 min) and sows (2.3±0.1 min). In experiment 2, 24 to 31 fresh morulae and/or blastocysts were transferred to each of 24 recipients. Seventeen animals (70.8%) farrowed an average of 6.9±0.7 piglets, of which 0.6±0.3 piglets were born dead. In conclusion, the procedure described in this study offers new possibilities to transfer embryos nonsurgically to the uterine horn of pigs.

Successful non-surgical transfer of horse embryos to mule recipients.

Mules, hybrids resulting from the mating of a horse mare (Equus caballus, 2n = 64) to a Jack donkey (E. asinus, 2n = 62), are generally infertile. Five horse embryos were transferred non-surgically to two cyclic and one acyclic recipient mules. In the mares and cycling mules, oestrus and ovulation were induced with, respectively, d-cloprostenol and human chorionic gonadotrophin (hCG). The acyclic mule, on the other hand, received oestradiol benzoate when the embryo donor was showing oestrus and progesterone after the donor had ovulated and until pregnancy diagnosis. Non-surgical embryo collections were attempted on day 7 after ovulation and recovered embryos were transferred transcervically into the mules' uteri. Mules that became pregnant were blood sampled serially for equine chorion gonadotrophin (eCG), progestagen and total conjugated oestrogen concentrations until around 6 months of gestation. The three embryos transferred to the acyclic mule did not produce any pregnancies whereas both embryos transferred to the cycling mules resulted in the birth of live foals. The peak concentration and duration of secretion of eCG differed markedly between the two pregnant mules, although both animals appeared to develop secondary corpora lutea beyond day 40 of gestation, as in normal intraspecies horse pregnancy. Moreover, the rise in serum oestrogen concentrations from around day 90 was also similar to that seen in normal pregnant mares. Parturition occurred spontaneously on day 348 of gestation in both mules and the resulting colt foals developed normally to weaning. Thus, cycling mules can carry a horse conceptus after non-surgical embryo transfer and give birth to a normal mature foal.

Recent developments in pig embryo transfer

Porcine embryo transfer has been performed for approximately 50 years, and surgical methods have proven to be reliable for collection and transfer of embryos. However, surgical collection and transfer have the disadvantage of being less useful on the farm. Recently, new procedures for both collection and transfer of embryos have been developed to improve usefulness. The surgical procedure has been refined to a minimally invasive procedure, using endoscopy for collection and transfer of embryos. A nonsurgical procedure for embryo collection has also been devised, but is limited to use in sows with surgically shunted (shortened) uterine horns. Nonsurgical embryo transfer procedures have been developed recently and have proven to be successful. The nonsurgical procedures are preferable to surgical procedures from an animal welfare point of view and because these procedures can be performed on farms without the need for special facilities.

Early embryo development in nuclear transfer clones derived from somatic cells of a high genetic merit dairy cow

AbstractThe reported post transfer survival rate to term of cloned embryos derived from both undifferentiated blastomeres and differentiated foetal and adult cells is very low (typically less than 20%). Furthermore, it is acknowledged that there are many technical issues that remain to be resolved to improve the efficiency of nuclear transfer before the technique will find widespread, practical and cost-effective use in multiplying valuable livestock in agriculture. The purpose of this study was to compare early embryo morphology following embryo transfer of nuclear transfer blastocysts derived from somatic cells of an adult Friesian cow with that of standard in vitro-produced embryos. In the present study, 150 embryos were transferred in bulk (i.e., 15, 20 or 25 per recipient) to the ipsilateral uterine horn of 8 recipients using standard non-surgical embryo transfer procedures. Embryos were then recovered following necropsy on either Day 14 or Day 23 of pregnancy and developmental traits described. Embryo recovery and elongation rates were similar on Day 14 of pregnancy (Table 1), although cloned conceptuses were longer and narrower (P&lt;0.05). Embryo recovery and viability rates by Day 23 were similar, although many of the developmental traits appeared more advanced in cloned conceptuses. (Table 1). Allantois development was different because of greater widths and the presence of ‘spurs’ that were not observed with in vitro-produced embryos. We conclude that apparently abnormal conceptus development occurs by about 3 weeks of pregnancy following nuclear transfer, but that embryo survival is not compromised at this early stage of development.

Reproductive biotechnologies: current status in porcine reproduction

During the past decade, considerable attention has been directed toward the development of reproductive technologies for both research purposes and for more controlled swine reproduction. Artificial insemination is an example of a technology that has continued to be expanded from early use in European countries to the USA and Canada where it is now estimated that a majority of the sows bred are artificially inseminated. In addition, several significant technological advancements have been made in the genetic modification of swine and interest has been generated in the possible use of swine as donors of specific tissues and of organs for the improvement of human health. At the same time, the systems for production of swine for human food continue to undergo major changes including, in some countries, the consolidation of swine into large, integrated units. These swine operations are very receptive to the use of technologies to reduce labor costs as well as a basis for increased production efficiency. Therefore, the combined interest in swine reproductive technologies by both the medical field and the swine industry creates an increased effort for the development of new technologies as well as for the implementation of existing ones. One of the more rapid technological advancements this decade has been the progress in in vitro production (IVP) of swine embryos. Major advancements have been made on the development of procedures for production of large numbers of embryos from oocytes collected at slaughter houses which are then matured (IVM) and fertilized (IVF) in the laboratory. Success in IVP has stimulated increased research in other areas that can be enhanced by the availability of embryos without a requirement for surgical collection from gilts or sows. One example is the combined use of IVF, gender-sorted sperm cells, and embryo transfer to produce offspring of a predicted sex. In a related area, instrumentation for non-surgical embryo transfer has recently been developed that results in significant improvement in this technology. Similar achievements have been gained in cryopreservation of embryos by vitrification. These developments will be reviewed with emphasis on the in vitro production of embryos from immature oocytes.

Effect of asynchronous non-surgical transfer of porcine embryos on pregnancy rate and embryonic survival

Embryo survival was determined after non-surgical transfer to recipients with a variable synchrony of ovulation. Groups of 10 to 15 freshly weaned multiparous sows (donors and recipients) were checked and paired for time of ovulation, resulting in recipients ovulating from 24 h before to 36 h after the donors (‘asynchrony’ of −24 to +36 h). Embryos were collected from 34 donors at 120 h (range 108–132 h) after ovulation and 16.6±2.4 morulae and blastocysts were transferred to 31 recipients. Pregnant recipients were slaughtered on Day 35 (Day 0=ovulation) to evaluate embryonic survival. Twelve recipients were pregnant at Day 21 and five were still pregnant at Day 35. One recipient was excluded due to cystic ovaries. An asynchrony of +18 to +36 h resulted in 1/12 recipients pregnant at Day 21 and no pregnancies at Day 35, while an asynchrony of −24 to +12 h resulted in 11/18 recipients pregnant at Day 21 and five still pregnant at Day 35 ( P<0.05). The presence of ≥6 morulae within a litter never resulted in pregnancies at Day 21 (0/9), while with <6 morulae, 12/21 recipients were pregnant at Day 21 ( P<0.05), irrespective of the degree of asynchrony. The results seem to indicate that only blastocysts should be transferred successfully by a non-surgical procedure at 108 to 132 h after ovulation. Recipients should ovulate between 24 h before to 12 h after the donors. Transfers to recipients ovulating 18 to 36 h after the donors appear to lead to very low pregnancy rates. However, these conclusions need to be confirmed in a study with more animals.

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5±11.7; n=48) than 1500 IU eCG (45.7±20.3; n=32; P<0.0001). Embryo recovery rate (82.9±16.9 %) and percentage expanded blastocysts (56.2±31.4 %) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (≤25 corpora lutea [CL]) or from superovulated sows (>25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5±11.5 μ) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3±15.3) than in the 1500 IU eCG group (70.2±1.9; P<.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71 % (10/14) and 46 % (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+3.0 and 11.1±2.7, and 14.2±7.0 and 10.5±4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 μm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43 % (6/14) and 77 % (10/13; Pš0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.

Ultrasonography as an important tool for the development and application of reproductive technologies in non-domestic species

Ultrasound imaging in reproductive sciences offers new opportunities regarding optimization of the induction of the sexual cycle and ovulation, superovulation regimes, contraception programs, semen collection and testicular sperm extraction techniques, ovum pick up and ovarian transplantation procedures, as well as the application of artificial insemination, embryo collection and transfer. In non-domestic species, most of which lack basic data, ultrasonography is an ideal tool to study reproductive biology in both captive and wild populations. The use of this imaging modality led us to develop new, or modify established, reproductive technologies. Ultrasonography has been an integral part of over 200 assisted reproduction procedures in 17 mammalian species performed by our research team between 1992 and 1999. These procedures included the initial characterization of sexual cycles, hormonal cycle induction, semen collection by electroejaculation or manual stimulation, non-surgical artificial insemination (AI), non-surgical embryo transfer and temporary hormonal contraception. For these investigations, a variety of newly developed equipment was applied and species-specific hormonal treatments designed. We used several commercial and customized ultrasound systems with a variety of technical features. Some relevant improvements of these applications will be described and the role of ultrasonography elucidated to.

Non-surgical Porcine Embryo Transfer by a Balloon Catheter Producing Subsequent High Farrowing Rate

The aim of this study was to improve a non-surgical technique for porcine embryo transfer that would be more effective and could be operated as easy as artificial insemination in the pig farm. Embryos were collected surgically from 13 gilts at 5 to 7 days post insemination by flushing uterine horns. A total of 144 embryos were immediately transferred into 11 recipients, 1 sow and 10 gilts, by the transcervical method with 2 types of balloon catheters (FA 361 and FA 1466 catheter, Fujihira Industries, Ltd., Tokyo). The recipient pigs were anesthetized and their heads laid down on the slope fastening their legs securely. After cleaning the external genitalia, the balloon catheter was inserted into the cervix through the vaginal speculum and fixed in the cervical canal by swelling the balloon. The number of embryos ranging from 5 to 26 per pig (Average: 13±6.8), of which developmental stages were morulae to hatched blastocyst, were transferred into the uterine horn through a catheter with a small volume of sterile physiological saline. Nine of 11 recipients (81.8%) got pregnant, while 2 of 9 pregnant pigs had an abortion at early time of gestation. The remaining 7 pigs farrowed a total of 25 live piglets (3.6±1.5). The embryonic survival rate was significantly (P<0.05) higher in the recipients transferred embryos by the catheter FA 1466 than in those by the catheter FA 361, although there was no significant difference in pregnancy rate. Moreover, in the catheter FA 1466-group, the number of young correlated closely with the number of transferred embryos (r=0.99, P<0.01). The results showed that the balloon catheter could be used for non-surgical transfer of porcine embryos, but the embryonic survival rate was affected by the type of catheter.

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo ( Bubalus bubalis) : A retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n=52) or induced (n=39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at ȡ12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL Size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P<.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.

Nonsurgical collection and nonsurgical transfer of preimplantation embryos in the domestic rabbit (Oryctolagus cuniculus) and domestic ferret (Mustela putorius furo)

The objective of this study was to develop nonsurgical methods of embryo collection and transfer in domestic rabbits (Oryctolagus cuniculus) and domestic ferrets (Mustela putorius furo) to serve as models for use in mammals in which surgical procedures are the usual means for applying embryo transfer technology. Specially designed transcervical catheters were used together with a fibre optic endoscope to visualize and then catheterize the rabbit and ferret cervices. Five consecutive transcervical uterine flushes in each of eight superovulated female rabbits 78-89 h after an ovulatory injection of LH resulted in the retrieval of 187 embryos, for an average of 23 embryos per rabbit. A total of 116 embryos were nonsurgically transferred to the uteri of ten recipients, and resulted in 23 young (20%). Eight rabbits (80%) produced young with an average litter size of 2.88 (range 1-7). Ten consecutive transcervical uterine flushes in each of 37 female ferrets 145-178 h after an ovulatory injection of hCG resulted in the retrieval of 324 embryos, an average of 8.76 embryos per ferret. A total of 251 embryos from 27 donors were nonsurgically transferred to the uteri of 31 recipients, and resulted in 65 young (26%). Twenty-eight of the recipients (90%) were initially pregnant, as indicated by postpartum necropsies, and twenty-two ferrets (71%) produced young. The average litter size was 2.95 (range 1-7). This is the first report of live births resulting from the nonsurgical collection of embryos from a donor followed by nonsurgical transfer of those same embryos to a synchronous recipient. The methods reported here can serve as models for use in other mammals in which direct visualization and manipulation of the cervix are not possible, and will be particularly useful in endangered species.

Plasma oestrone and oestradiol concentrations throughout gestation in cattle: relationship to stage of gestation and fetal number

The objectives of the study were to characterise the peripheral plasma oestrone (E1) and oestradiol-17β (E2) concentrations throughout gestation in the cow and to correlate this with the stage of gestation and fetal number. Cows (n = 10) were equally divided into two groups after non-surgical embryo transfer of in-vitro matured and in-vitro fertilised (IVM-IVF) embryos; Group 1 received a single embryo, Group 2 received twin embryos. Blood was collected about every third day from day 0 (day 0 was defined as first day of standing oestrus), then daily for the last 10 days of gestation and sampling was stopped one day post partum. Plasma E1 concentration exceeded that of E2 throughout gestation in both groups of cows. The time trend concentrations of plasma E1 were significantly affected by the stage of gestation (P < 0·01) and fetal number (P < 0·01) in the last two trimesters of gestation. The time trend concentrations of plasma E2 were significantly affected by the stage of gestation (P<0·01) but not foetal number (P = 0·09). In both groups there was marked preparturient increase in E1 and E2 concentrations. Plasma E2 profile between days 10 prepartum to parturition paralleled E1 in cows carrying a single foetus but was disparate during the same period in the twin-bearing cows.To conclude, our results indicate that although plasma E1 concentration was greater than E2 throughout gestation, both were related to the stage of gestation and that fetal number was correlated with circulating E1 levels in the last two trimesters of gestation.

Effect of eCG dose on embryonic survival in the sow after non-surgical embryo transfer

Effect of eCG dose on embryonic survival in the sow after non-surgical embryo transfer