Successful nonsurgical embryo transfer in buffalo (Bubalus bubalis ) in Bulgaria

Forty-one Day 5.0 to Day 5.5 embryos and one unfertilized ovum were recovered nonsurgically from 24 superovulated, parous buffalo (Bubalus bubalis ) and transferred nonsurgically to 28 synchronized recipients by a team of Bulgarian and American scientists. Five pregnancies were established and four live buffalo calves were born at the end of normal gestation periods.     

一种非手术性小鼠胚胎移植技术

为了提高非手术性小鼠胚胎移植技术的成功率,利用塑料移植导管模拟非手术胚胎移植过程,通过观察染料在子宫角的分布而评估胚胎移植效果,并在此基础上将自然妊娠3.5 d的小鼠囊胚经子宫颈移植受体小鼠。结果表明:将CD-1小鼠囊胚移植假孕2.5 d小鼠单侧子宫角,平均70.9%的胚胎能够发育至成活新生仔鼠,建立了高效非手术性小鼠胚胎移植技术。该方法简便快捷、不易污染、费用低,无需专业的手术器械,且符合实验动物伦理原则,完全可以取代手术法胚胎移植技术,更重要的是,它为人类和其他大动物的胚胎移植提供了研究模型。     

A simple technique for nonsurgical embryo transfer in mice

A simple method was developed for nonsurgical transfer of mouse embryos which enabled transfer to both uterine horns. Embryos were picked up in a modified capillary tube and transferred through the cervix into each uterine horn of an unanesthetized mouse. A glass speculum was used to facilitate location of the cervix. The technique was found to be as successful (up to 60% of embryos transferred developed to term) as surgical methods yet was simpler and eliminated surgical trauma to the recipient mouse.     

A procedure for successful nonsurgical embryo transfer in swine

The current study was undertaken to develop a successful procedure for the nonsurgical transfer of pig embryos. A total of 663 embryos were surgically collected on Day 4 or 5 from 55 donors, of which 542 embryos of acceptable quality were nonsurgically transferred to 46 recipients. Nonsurgical recipient gilts were sedated 15 min prior to transfer with 20 mg im acepromazine maleate. A disposable insemination spirette with an attached 3-way stopcock was manipulated into the cervix of each gilt. Embryos were expelled from a tomcat catheter into the spirette, and 10 to 12 ml of Whitten's medium were used to flush embryos through the spirette into the reproductive tract. Sixteen (34.8%) recipient gilts did not return to estrus before Day 36, and 10 (21.7%) gilts farrowed with an average litter size of 4.3 +/- 0.7. Embryos were collected from an additional 20 donors and were surgically transferred to an additional 19 recipients. Surgical transfers conducted at the same time as the nonsurgical transfers resulted in 12 (63.2%) gilts farrowing and 7.1 +/- 0.6 pigs were born per litter. In conclusion, a procedure has been developed for nonsurgical transfer of swine embryos which simplifies the process of embryo transfer and which may increase the potential for utilization of embryo transfer technologies by swine producers.     

A study of nonsurgical embryo transfer in the mare

The success rate of nonsurgical embryo recovery was influenced neither by year nor by season within years. The preferred method of nonsurgical embryo transfer was by Cassou pistolette. From a total of 15 attempts to transfer embryos nonsurgically, 9 (60%) were successful. Of the five attempts during February through April 1982, only one was successful in producing a live foal. The degree of synchrony between the ovulations of the donors and recipients in these five attempts ranged from +3 to -3 d. The recipient of the successful transfer ovulated on the same day as the donor. Eight of the ten attempts during September through December 1982 produced live foals. Synchronization of ovulations between the donors and recipients in these transfers ranged from 0 to -2 d. Repeated attempts to recover embryos had no deleterious effects on fertility of the donors.     

Successful recovery of preimplantation embryos by nonsurgical uterine flushing in the bonnet monkey

A major limitation to progress in primate embryology is the lack of an adequate supply of preimplantation embryos. We describe a method for recovering preimplantation-embryos in bonnet monkeys (Macaca radiata ) using a nonsurgical uterine flushing technique similar to the one previously employed in rhesus monkeys. Forty cyclic females were screened for cervical cannulation, and 10% of these had an impassable cervix. Eleven females suitable for cannulation were selected, and 27 menstrual cycles were monitored over a 5-mo period. Seventy-one percent of the cycles showed estrogen peaks, which were observed between Days 9 and 14 of the cycle. Following natural mating, uterine flushings were performed on Days 5 to 8 of pregnancy (Day 0 = the day following the estrogen peak). Of the 27 recovery attempts, 9 (33.3%) resulted in the recovery of ovulation products, including those of an unfertilized oocyte and empty zona (2 cases), retarded cleavage-stage (4 to 8-cell) embryos (4 cases), morula (1 case) and blastocysts (2 cases). These results show, for the first time, that the nonsurgical uterine flushing technique can be successfully performed to recover uterine-stage preimplantation embryos from bonnet monkeys.     

Factors affecting the success of surgical and nonsurgical equine embryo transfer

Nonsurgical embryo recovery was attempted from light-horse and draft mares. Embryo recovery rates were not affected (P>.05) by technician or stallion but were lower (P<.05) from draft mares (44%) than light-horse mares (67%). Sham transfer of embryos on day 8 post-ovulation did not (P>.05) increase the number of mares returning to estrus by 22 days post-ovulation. Method of embryo transfer greatly affected pregnancy rates. Embryos transferred surgically during March–June resulted in 0 of 12 pregnancies versus 13 of 25 pregnancies obtained during July–September, This strongly suggests a seasonal influence on pregnancy rates. Technician influenced (P<.05) the success of nonsurgical transfer (46.2% vs. 7.7%). In addition, protection of the insemination rod with a sheath (guarded method) appeared to provide some advantage over an unguarded method of nonsurgical transfer (54% vs. 23%). Lastly, a preliminary experiment was conducted to evaluate transfer of embryos via flank incision. Four of 5 embryos transferred by this method resulted in a pregnancy at 50 days post estrus.     

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 (< or = 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 microm) 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<0.05) 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 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), 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.     

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 superovulation, nonsurgical collection and transfer of embryos from Brahman cows

Nonsurgical recoveries and transfers of embryos were performed at the McKellar Embryo Transplant Center from 122 superovulated Brahman cows. FSH-P (Armour) was used to superovulate all cows at dose levels ranging from 36 to 48 mg total FSH-P. Luteal regression was induced by use of 40 mg PGF(2(alpha)) in all 122 cows. Embryos were transferred into recipients 6, 7 or 8 days after observed estrus. Embryos were successfully collected from 82% of the FSH-P treated cows. The dose level of FSH-P affected numbers of embryos collected (P<.05). Numbers of embryos collected from cows superovulated with 36-38, 40, 42, 43, 44, 45, 46 and 47-48 mg FSH-P were 2.8 +/- 1.0, 6.8 +/- 1.1, 9.4 +/- 1.4, 10.0 +/- 2.7, 7.1 +/- 1.6, 6.8 +/- 2.0, 5.0 +/- 1.7 and 4.6 +/- 2.0 embryos, respectively. The dose level of FSH-P also affected numbers of embryos transferred (P<.10). Number of embryos transferred from cows superovulated with 36-38, 40, 42, 43, 44, 45, 46 and 47-48 mg FSH-P were 2.8 +/- 1.9, 5.2 +/- 0.9, 6.9 +/- 1.2, 6.7 +/- 2.1, 4.8 +/- 1.3, 5.1 +/- 1.4, 3.4 +/- 1.2 and 3.2 +/- 2.1 embryos, respectively. The developmental stage (D) of the embryo was also a factor in pregnancy rate of recipients (morula = 13.8%, blastocyst = 22.1% and expanded blastocyst = 29.9%; P<.005). The skill of the technician (T) transferring the embryo had a dramatic effect upon subsequent pregnancy rate of the recipients (T 1 = 46.0% vs T 2 = 22.6% pregnancy rate; P<.005). Pregnancy rate of recipients was also affected by the stage postestrus (S) at which the embryo was transferred (day 6 = 23.5%, day 7 = 25.5% and day 8 = 42.3% pregnancy rate; P<.05). Interactions were found between T x S, T x D, S x D and T x S x D (P<.05). These data indicate that use of 40, 42, or 43 mg total doses of FSH-P were quite effective in superovulating the Brahman cow. Recipients transferred on day 8 postestrus achieved higher pregnancy rates than recipients transferred on days 6 or 7 postestrus. Embryos transferred in the expanded blastocyst stage of development proved to yield the highest pregnancy rates in recipients.     

Successful embryo transfer in Tianzhu white yak using standard protocol

The present study was carried out to investigate the efficiency of superovulation, oestrus synchroni-zation, and embryo recovery in Tianzhu white yaks and also to confirm the pregnancy rate of black yaks, to which embryos collected from white yaks were transplanted. Forty-seven yaks were selected from different experiment groups, including 10 Tianzhu white female yaks (donor, group A) and 37 black female yaks (recipient, groups B and C). Superovulation of the donor was induced by the application procedure of CIDR-B FSH PG. Oestrus synchronization of recipients was induced using two meth-ods: group B was given the same treatment as group A, except that the follicle-stimulating hormone (FSH) injection was not administered, whereas group C was injected with cloprostenol only once when corpus luteum (corpora lutea) was (were) palpated. The results showed that the oestrous rates in group A were higher (80%) than those in group B (60%) and group C (44.5%). As for the efficiency of su-perovulation, it was indicated that the mean numbers (±SD) of total corpora lutea, follicles, viable (transferable), and degenerated embryos were 4.75 ± 2.19, 1.13 ± 0.83, 2.50 ± 1.31, and 1.38 ± 0.92, re-spectively. The mean embryo recovery rates were 55.6%. All together, 18 viable embryos of Tianzhu white yak were obtained and 12 of them were transplanted to 10 recipients. The pregnancy rate was 50% and the delivery rate was 40%.     

Successful embryo transfer in Tianzhu white yak using standard protocol

The present study was carried out to investigate the efficiency of superovulation, oestrus synchronization, and embryo recovery in Tianzhu white yaks and also to confirm the pregnancy rate of black yaks, to which embryos collected from white yaks were transplanted. Forty-seven yaks were selected from different experiment groups, including 10 Tianzhu white female yaks (donor, group A) and 37 black female yaks (recipient, groups B and C). Superovulation of the donor was induced by the application procedure of CIDR-B + FSH + PG. Oestrus synchronization of recipients was induced using two methods: group B was given the same treatment as group A, except that the follicle-stimulating hormone (FSH) injection was not administered, whereas group C was injected with cloprostenol only once when corpus luteum (corpora lutea) was (were) palpated. The results showed that the oestrous rates in group A were higher (80%) than those in group B (60%) and group C (44.5%). As for the efficiency of superovulation, it was indicated that the mean numbers (±SD) of total corpora lutea, follicles, viable (transferable), and degenerated embryos were 4.75 ± 2.19, 1.13 ± 0.83, 2.50 ± 1.31, and 1.38 ± 0.92, respectively. The mean embryo recovery rates were 55.6%. All together, 18 viable embryos of Tianzhu white yak were obtained and 12 of them were transplanted to 10 recipients. The pregnancy rate was 50% and the delivery rate was 40%.     

Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys

Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds.     

The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer

This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2–6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry.     

Successful embryo transfer in a small dasyurid marsupial, Sminthopsis crassicaudata

A technique for the transfer of early embryos from the uterus of donors to recipient females of a small dasyurid marsupial, Sminthopsis crassicaudata , is described. When the recipients were killed 3 to 5 d later, a few expanded blastocysts were found to be present in the uteri of most of the females provided that corpora lutea were present in the ovaries. This study demonstrates successful embryo transfer in a small marsupial species and supports the view that secretion of the endometrium due to luteal activity may be necessary for development of the expanded unilaminar blastocyst stage.     

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.     

A device for nonsurgical transfer of bovine embryos and its effect on uterine contamination

A simple, inexpensive, concentric cannula device is described for the transfer of embryos through the cervix in cattle. Its effectiveness in minimizing the introduction of contamination into the uterus was tested by bacteriological surveillance of the various components after use, and of the uterus of 12 cows slaughtered 2 – 3 days after sham transfer. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in 100%, 66% and 50% respectively. Bacteria were always found in the samples obtained from the vulva and vagina, but in only 55% of those taken from the external os of the cervix. Pregnancy resulted in $\text{5}\text{10}$ cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum.