Non-surgical embryo transfer and live birth in a llama

The uteri of two donor female llamas were flushed non-surgically and one viable 7-day-old embryo was recovered. The embryo was transferred non-surgically within four hours into a recipient female llama whose estrous cycle was synchronized with the donor by injection of gonadotropin releasing hormone (GnRH). Pregnancy was confirmed 20 days after transfer, at which time the level of serum progesterone was determined and found to be consistent with that of gestation (>/=100 ng/100 ml). A normal and healthy male weighing 33 1bs was born on October 8, 1984, 326 days following transfer of the embryo to the recipient.     

Hepatic biotransformation in farmed blue fox and raccoon dog

In farmed raccoon dogs and blue foxes, the hepatic content of cytochrome P-450 and the activity of polysubstrate mono-oxygenase with benzo(a)pyrene and 7-ethoxycoumarin as substrates, as well as activity of UDP glucuronosyltransferase were of the same order of magnitude as those in the laboratory rat. The amount of reduced glutathione tended to be higher in canids. There existed differences in the biotransformation activities between raccoon dogs from different farms. These cannot, however, be systematically related to the quality of food, body composition and succinate dehydrogenase activity.     

Nonsurgical embryo transfer in the scimitar-horned oryx (Oryx dammah): Birth of a live offspring

The objective of this project was to determine if modifications of methods of estrous synchronization, superovulation, embryo recovery, and transfer used successfully in other ungulates, both domestic and nondomestic, could be applied to scimitar-horned oryx (Oryx dammah). Donors were two parous females and recipients were one parous and two nulliparous females that were given a total of two cloprostenol injections at an interval of 0 and 13 or 12 days, respectively. Donors were treated with follicle-stimulating hormone (FSH-P, Schering, Kenilworth, NJ) b.i.d. for 4 days and placed with a fertile male. Seven days after the last FSH-P injection, nonsurgical uterine lavages were performed on both donors. One good-quality embryo at the morula stage was recovered and nonsurgically transferred into the right uterine horn of the parous recipient. A healthy female calf born at 247 days post-transfer represents the first known live birth of scimitarhorned oryx following embryo transfer. These results provide additional evidence that estrous synchronization and embryo transfer techniques used in other ungulates can be applied to endangered antelopes such as the scimitar-horned oryx.     

Successful embryo transfer in the silver fox (Vulpes vulpes)

Surgical embryo transfer in the silver fox was investigated as part of a larger project concerning the conservation of endangered canine species using modern artificial reproduction techniques with the farmed fox as a model. The animals were chosen on the basis of synchrony in natural oestrus. The timing of ovulation and artificial insemination was determined by measuring electrical resistance in the vagina. Twenty-nine embryos were flushed from eight humanely killed donor females and transferred surgically into the uteri of eight recipients. One recipient female gave birth to two male pups 47 days after the transfer of four expanded blastocysts and one embryo at the 16-cell stage derived from a donor female flushed 10 days after artificial insemination.     

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 0.2 kg 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.     

Clock gene Bmal1 in mice embryo is dispensable for early embryo development but critical for live birth

In adult animals, the significance of circadian clocks in the regulation of physiology is well established. However, the physiological roles of embryonic clock genes on early embryo development, implantation and perinatal survival are still unclear. In the present study, using genotyping, embryo culture and transfer, the early embryo development, implantation, and perinatal survival of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo were studied. At cleavage stage, the genotype ratio of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo was 1:1.97:0.95, respectively (p > 0.05). Morula or early blastocyst developmental ratio was 83.8 ± 14.3, 87.1 ± 9.2 and 88.7 ± 14.5%, respectively (p > 0.05). After transferring of the three types of embryos to pseudopregnant wild-type mice, the implantation sites 4 days later was 7.7 ± 0.9, 7.2 ± 1.2 and 7.5 ± 0.5 (n = 4, F = 0.265, p = 0.773). Mean litter size of the mice after transferring with the three types of embryos was 5.5, 6.0, and 3.0 (n = 3, F = 30.3, p = 0.001). The development of Bmal1 null embryos was not impaired in preimplantation and early implantation stages, but the litter size had a trend to decrease.     

Surgical transfer of in vivo produced farmed European polecat (Mustela putorius) embryos

Surgical embryo transfer of farmed European polecat (Mustela putorius) was investigated as part of an ex situ preservation project. The long-term objective of the project is to develop effective technology for ex situ conservation of the European mink (Mustela lutreola), which is a highly endangered aboriginal European species. Twenty European polecat females, which served as a model species for the European mink, were humanely killed 4-9 days after first mating and embryos were recovered from oviducts and uteri. Donor-recipient pairs (n = 16) were generated by mating the donors (n = 20) once a day for 2 consecutive days with fertile males and by mating the corresponding recipients (n = 16) on the same days with vasectomized males. An embryo recovery rate of 70% (200 recovered embryos/284 corpora lutea) was achieved from 20 donors. Morulae and blastocysts were recovered between Days 5 and 9 after first mating and were regarded as the best developmental stages for uterine embryo transfer. A total of 172 embryos were transferred surgically under general anaesthesia into the ovarian third of the left uterine horn of 16 recipients with a thin glass capillary. Eleven recipients (69%) produced 72 pups equivalent to an average success rate of 42% (72 pups/172 transferred embryos). The average litter size was 4.5 (range 0-9). These results with this model species, farmed European polecat, demonstrate the potential of embryo transfer as an effective method for the preservation of the endangered European mink (M. lutreola). These species are closely related and have a similar reproductive physiology. However, success of applying embryo transfer in conserving European mink is still dependent on further studies both into its reproductive physiology and developing of improved flushing techniques for anaesthetized donors and the successful transfer of frozen-thawed embryos.     

First successful transfer of cryopreserved feline (Felis catus) embryos resulting in live offspring

Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.     

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.     

First live offspring born in superovulated sika deer (Cervus nippon) after embryo vitrification

The rapid growth in sika deer (Cervus nippon) farming and interest in their conservation is an impetus for development of embryo transfer (ET) procedures. However, a paucity of research has prevented widespread application of ET in this species. The objective of the present study was to establish a multiple ovulation and ET procedure with both fresh and vitrified embryos in sika deer. Multiparous weaned hinds (N = 18) were used as embryo donors during the reproductive season of 2008 at a local breeding farm in China. Estrus was synchronized in donors and recipients (N = 38) by inserting a controlled internal drug release for 12 days (insertion = Day 0). Superovulation was induced with a total of 320 mg of NIH-FSH-P1 (Folltropin-V; Bioniche, Belleville, ON, Canada) given as 40 mg im every 12 h from the afternoon of Day 9 to the morning of Day 13. After estrus was detected, donors were artificially inseminated using a transcervical technique. The embryo recovery rate was 76.8% (63/82), including 1.6% (1/63), 77.8% (49/63), and 1.6% (1/63) blastocysts, morula, and eight-cell embryos, respectively. After transfer of fresh and vitrified embryos, pregnancy rates were 85.7% and 61.6% and birth rates were 64.3% and 53.9% (P > 0.05). In conclusion, we developed a satisfactory multiple ovulation and ET procedure in farmed sika deer using vitrified embryos.     

Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.     

Successful culmination of pregnancy and live birth following the transfer of frozen-thawed buffalo embryos

A total of 141 embryos was recovered by nonsurgical flushing of the uterus of 31 superovulated buffalo. A total of 66 good quality embryos (Grade I and Grade II) was frozen using 1.4 M glycerol. Forty-two of the frozen embryos were thawed randomly over a 1-year period, and a total of 39 embryos (Grades I, II or III post thaw) were transferred into an equal number of estrus synchronized recipients. Of 11 confirmed pregnancies, 9 calves were born live.     

Non-invasive faecal steroid monitoring of ovarian and adrenal activity in farmed blue fox (Alopex lagopus) females during late pregnancy, parturition and lactation onset

In the semi-domesticated blue fox, handling stress may influence reproductive performance and increase perinatal pup loss. Ovarian and adrenal steroids were analysed in faecal samples collected from mid-gestation through the first week of lactation in 40 female blue foxes to characterize hormone patterns during this important reproductive period. Daily faecal samples were collected from 40 foxes during 30 pregnancies, one late abortion and nine bred-matched non-pregnancies. Mean concentrations of faecal progestagens over the 10 days before birth were significantly higher in pregnant compared to non-pregnant females (51+/-1.50 microg/g versus 36+/-3.72 microg/g, respectively; P < 0.01). From 10 to 3 days before whelping, total faecal oestrogen concentrations also were higher (P < 0.01) in pregnant (1082+/-41.69 ng/g) than non-pregnant (628+/-72.43 ng/g) foxes, before declining to non-pregnant values (402+/-24.88 ng/g) after parturition. Overall mean faecal corticoid concentrations from 3 to 20 days before whelping differed between pregnant and non-pregnant foxes (128+/-3.11 ng/g versus 103+/-5.86 ng/g, respectively; P < 0.01). Furthermore, in pregnant foxes, corticoid excretion increased further from 2 days before to 3 days after whelping (216+/-13.71 ng/g; P < 0.01). Thereafter, corticoid concentrations were similar between pregnant and non-pregnant females (P > 0.05). In sum, the faecal steroid hormone patterns for oestrogens and progestagens were similar to those previously obtained by analyses of fox serum hormones, with both steroids being higher in pregnant than non-pregnant foxes at the end of gestation. The elevation in corticoid concentrations in pregnant females suggests that adrenal activation is involved in the initiation of parturition in the blue fox. Thus, faecal steroid analyses can be used to monitor ovarian activity during pregnancy and pseudopregnancy in farmed blue fox females.     

Preferences of farmed blue foxes for different floor types

In the first experiment, farmed blue foxes were allowed to choose for 1 week between four standard farm cages equipped with different floor materials: plastic-coated wire mesh, dry wood, dry sand and wet (summer) or icy (winter) sand. Resting consisted of 10-15 separate bouts/day occupying 50-60% of the total 24-h. There were no other differences in the use of the cages except that the time spent on, and the duration and number of resting bouts were lower on wet or icy sand, resting periods being more affected than activity. In the second experiment, two cages were connected with a 1.2 m tunnel. One cage was always elevated (50 cm) compared to that one which was lower. One cage of each pair had a wire floor whereas the other cage either had sand or wire floor. Sand floor was preferred for active behaviours and wire floor for resting if these were on elevated level. Of two identical wire-floored cages, the elevated one had the priority. Foxes preferred to rest on the same floor where they had finished their previous resting bout, often exclusively and independently of floor material or floor level.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

Pregnancy and live birth from nonsurgical transfer of in vivo- and in vitro-produced blastocysts in the rhesus monkey

Embryo transfer in the rhesus monkey has been historically limited to transfer of cleavage stage embryos. In order to allow genetic manipulation of rhesus embryos in vitro, without using invasive surgical techniques, it is important to explore the transfer of morula and blastocyst stage embryos. Embryos were produced by in vitro fertilization from gonadotropin-stimulated monkeys, or were obtained by nonsurgical uterine flushing of naturally mated or artificially inseminated females. Nonsurgical transfer was accomplished by inserting a metal guide through the cervix into the uterus, after which a hollow cell sampler was inserted over the guide. The guide was removed and a catheter was inserted containing one to five embryos. Several pregnancies resulted from in vitro- and in vivo-derived blastocysts, and two pregnancies were carried to term resulting in one live birth. Blood samples were collected regularly to monitor plasma levels of chorionic gonadotropin, luteinizing hormone, and progesterone. The recipients received progesterone as a subcutaneous implant or daily injections from the day of transfer. The approach described in this study provides the opportunity to explore transgenic and chimeric models in the monkey by the development of noninvasive methods to transfer late-stage embryos that have been manipulated in vitro.     

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.     

Development of rabbit zygotes into blastocysts in defined protein-free medium and offspring born following culture and embryo transfer

We report here on an improved, completely defined culture system for producing embryos in vitro which mimics development in vivo. This system avoids the confounding effects of the many unknowns introduced by the multivariate components of the serum or by unknowns attached to bovine serum albumin (BSA). Zygotes were obtained from superovulated rabbits and cultured in modified defined RPMI 1640:Dulbecco's MEM, 1:1 (RD) medium. The effect of a novel and potentially ideal antioxidant, tempol, was tested (20 to 0.001 mM) but found to be either toxic or ineffective. In the presence of 20% O(2), 600 units of Superoxide dismutase or 2.5 mM of taurine increased embryo hatching after 72 h of culture in RD medium to 75 and 76%, respectively, compared with 46% in the control (P < 0.05). The need for antioxidants was reduced with 5% O(2). The beneficial effects of RD medium were demonstrated when 60 zygotes cultured for 48 h to the early blastocyst stage in this medium were transferred and resulted in 30 young (50%) compared with 35/60 (58%) young from uncultured control transfers. Only 12% of the young were obtained from slower developing morulae. Thus, high viability was established for rapidly growing embryos in culture, but fewer slow growing embryos survived after transfer. A further comparison of embryos cultured in RD medium with a high potassium simple, optimized, defined medium (KSOM), revealed that both yielded results approaching those of direct transfer without culture. Simple defined media may also be useful for the culture of embryos of other species.