Clinical implications of developments in in vitro fertilisation.

During February 1979 to December 1983, 831 infertile couples were treated by in vitro fertilisation and embryo transfer. The problems they faced included deciding on the number of oocytes to be collected at laparoscopy, the numbers to be donated or fertilised, the numbers of embryos to be transferred and frozen, and whether abnormal embryos should be used for research or discarded. The 831 patients received a total of 1530 treatment cycles. Of the 763 patients for whom complete data were available, 136 (17.8%) became pregnant. The rate of pregnancy, however, increased dramatically from 7.4% when only one embryo was transferred to 21.1% and 28.1% when two and three embryos were transferred, respectively. The chance of multiple pregnancy also increased with the number of embryos transferred, but the risk (2% for twins) was far outweighed by the relatively poor result after transferring a single embryo. Out of 40 embryos freeze-thawed, 23 survived thawing and were transferred; of these, 4 (17%) resulted in pregnancy. Thirty four transfers of donor oocyte embryos also resulted in four pregnancies (12%), but two of these ended in abortion. Neither microscopy nor any other available test can determine the potential of an oocyte to result in pregnancy, so that discarding oocytes that may look abnormal simply reduces the chances of conception--both for the patient and for any prospective recipient of donor oocyte embryos. In any case, abnormal embryos tend to die when growth is allowed to continue in vitro. Probably all oocytes harvested from a patient should be inseminated and the utilisation of the embryos decided once the number developed is known.     

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.     

Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro

The developmental capacity of reconstructed bovine oocytes that contained nuclei from growing stage oocytes, 70-119 microm in diameter, was assessed after fertilization in vitro. Nuclei from growing stage oocytes of adult ovaries were transferred to enucleated, fully grown germinal vesicle (GV) stage oocytes. After culture in vitro, the reconstructed oocytes matured, forming the first polar body and MII plate. To supply the ability to form pronuclei, the resultant MII plate was transferred to enucleated MII oocytes, which were obtained by in vitro culture of cumulus-oocyte complexes. After fertilization in vitro, 11-15% of the reconstructed oocytes developed to morulae and blastocysts. To assess the ability to develop to term, a total of 27 late morulae and blastocysts were transferred to 19 recipient cows. Of the three cows that subsequently became pregnant, one recipient, who received two embryos derived from reconstructed oocytes with a nucleus from oocytes 100 to 109 microm in diameter, continued the pregnancy to Day 278 of gestation. This pregnancy, however, was unexpectedly a triplet pregnancy that included a set of identical twins and resulted in the premature birth of the calves, followed by death from lack of post-parturient treatment. These results show that bovine oocyte genomes are capable of supporting term development before the oocytes grow to their full size, which suggests that growing stage oocytes can be directly used as a source of maternal genomes.     

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs

We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary, oviduct and uterus, clear view of the organ manipulation without exposure and exteriorization of viscera during surgery.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Superovulation, collection and transfer of embryos and demi-embryos from Boran(Bos indicus ) cows and heifers

Twenty-three Boran(Bos indicus ) cows and heifers were superovulated with pregnant mare serum gonadotropin (PMSG); a total of four embryos and 4.1 +/- 0.3 (mean +/- SEM) ova per ova-producing donor resulted. Follicle stimulating hormone (FSH-P) was then used to superovulate 49 Boran cows for a total of 106 superovulations, of which 63 (59.4%) produced an average of 3.7 +/- 0.4 (mean +/- SEM) embryos. The embryo production was not influenced by either the season or the number of times(one to five) the cows were superovulated. A higher pregnancy rate was obtained when the selection of Boran recipients was based on their plasma-progesterone values (overall 52.5%, single embryos 63.3%, twin demi-embryos 45.8%) than when they were selected by palpation per rectum only (overall 43.8%, single embryos 50%, twin demi-embryos 36.4%). The twinning rate of twin demiembryos was 62.5%, whereas only single calves were born after transfer of two embryos per recipient. No pregnancies were produced following transfer of twin demi-embryos without zonae pellucidae. Transferring single demi-embryos gave a low pregnancy rate (13.3%). Twelve donor Boran cows (21 superovulations) bred with their fathers resulted in a high rate of early embryonic death; additionally, only 20.9% (overall) of the recipients became pregnant. Estrus synchronization of Boran cows with a progesterone releasing intravaginal device (PRID) for a short period (7 d) combined with one injection of prostaglandin (Day 6) produced a larger number of good quality recipients (70.5%) than using double prostaglandin injections (60%).     

Farrowing rates and litter size following transfer of vitrified porcine embryos into a commercial swine herd

The objective was to determine farrowing rates and litter sizes that could be achieved in a typical farm-to-farm porcine embryo transfer program using vitrified blastocysts that were zona pellucida intact when cryopreserved. The embryos were transferred surgically on-farm into recipient sows that were managed throughout gestation and farrowing under the same conditions as other sows in the herd. Twenty recipient sows (mean parity 2.1) received a total of 568 embryos; seven received 203 embryos derived from donor sows, five received 139 embryos from gilts and eight received a mixture of 161 embryos from sows and 65 from gilts. Sixteen sows (80%) were confirmed pregnant at approximately 35 days gestation, 15 farrowed at full term (farrowing rate 75%). One sow died during gestation (with a total of 18 fetuses in utero). A total of 123 piglets were born (mean, 8.2), of which 115 were born alive (mean, 7.7). Of the 568 embryos transferred to all 20 sows, 21.6% resulted in piglets born and 29.0% survived to produce piglets in sows that farrowed. There were no significant differences in embryo survival among sow, gilt or mixed sow and gilt embryos. The ratio of males to females was 71/52 and the mean birth weight was 1.6 kg (range 0.6-2.6 kg). In conclusion, vitrified zona pellucida intact embryos can be used to transfer genetic material from farm-to-farm with acceptable reproductive performance.     

Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.     

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF_2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0-3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.     

The production of mixed-species Bos taurus-Bos indicus twin calves

Bos taurus-Bos indicus twin calves were produced by transferring Day 6–8 Bos indicus or Bos taurus embryos to previously inseminated Bos taurus or Bos indicus cows. Embryos were transferred to 112 recipients of which 64 (57.1%) were diagnosed as pregnant by rectal palpation at 7–8 weeks gestation. Twin foetuses were diagnosed in 38 (65.5%) of 58 cows in which the location and number of foetuses was recorded. Substantial foetal losses occurred after 7–8 weeks and only 44 cows calved giving 21 sets of twins and 23 singletons. Six of the single calves were from transferred embryos. The duration of gestation of the twin pregnancies (282 days) was similar to that of single Bos taurus pregnancies (282 days for Jerseys and 286 days for Friesians) and 14 days shorter than single Bos indicus pregnancies (296 days). Consequently Bos indicus members of mixed-species twins were premature at birth and required intensive care to ensure their survival.     

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。     

云南省奶牛胚胎移植试验

实验用12头黑白花奶牛（3-9岁）,PGF[2α]作同步发情处理后,周期第10-12天,总剂量36 mg的FSH-P按递减方式每日肌肉注射2次,间隔12小时,共注射4天,在第3天加注PGF[2α]35 mg,进行超数排卵处理。12头供体牛共得胚135枚,可用胚数118枚,为总胚数的87.47%。平均每头得胚11.25枚。胚胎分别移植给20头同步发情处理的黑白花奶牛和20头黄牛受体（有3头为自然发情）,每头移植1枚胚胎。结果,奶牛受孕率为35%（受孕7头,产犊7头）;黄牛受孕率为40%（受孕8头,流产2头,产犊6头）。奶牛和黄牛总受孕率为37.5%。     

Non-surgical embryo transfer in cattle

Embryos collected surgically from donors superovulated with PMSG and synchronized with either prostaglandin F(2)alpha or progestagen impregnated sponges were transferred non-surgically to prostaglandin or progestagen synchronized recipients. One embryo was transferred to the uterine horn ipsilateral to the corpus luteum either through a flexible catheter introduced through a steel tube and passed to the uterine tip, or through a Cassou inseminating gun passed approximately 6 cm into the horn. Of 16 recipients receiving 5 or 6 day old embryos through the catheter (1976), 6 (38%) were palpated pregnant at 42 days and 4 (25%) subsequently calved. Of 16 recipients receiving 7 or 8 day old embryos through the straw and 16 through the catheter (1977), 10 (63%) and 3 (19%), respectively, were palpated pregnant (P<0.05) and 8 (50%) and 3 (19%), respectively, had normal embryos at slaughter 4 to 29 days after palpation (P reverse similar0.10 ). Forty 7 to 9 day old embryos were transferred through the straw in 1978. Eighteen (45%) of the recipients were palpated pregnant and 16 (40%) had normal embryos at slaughter 98 to 168 days after palpation. The success of the transfers in 1978 was affected by embryo quality [good vs poor embryos; 64% vs 22% recipients pregnant (P<0.01) and 59% vs 17% embryos surviving to slaughter (P<0.05)]. Also, in 1978, pregnancy rate was affected by the time taken to transfer the embryo with the highest rate achieved with the fastest transfers (P<0.10, b = -0.47). Injection of Indomethacin near the time of transfer, synchronization between donor and recipient onset of estrus and embryo age did not affect pregnancy rates. The pregnancy rate achieved after the transfer of good quality embryos by the straw technique was equal to that expected from surgical techniques.     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

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.     

Induction of twinning in Holstein and Japanese black cows by ipsilateral frozen embryo transfer

Induction of twinning by ipsilateral nonsurgical transfer of two frozen-thawed Japanese Black bovine embryos to each of 20 Holstein and 26 Japanese Black cows, that had been kept under a stable in private farm conditions, was examined. The cows were monitored every 20 days from Day 25 to Day 65 of gestation for pregnancy and fetus survival (estrus is Day 0). Seventy-five per cent (15 of 20), 65.0% (13 of 20) or 60.0% (12 of 20) and 61.5% (16 of 26), 53.8% (14 of 26) or 50.0% (13 of 26) of Japanese Black cows were diagnosed pregnant at 25, 45 and 65 days after transfer by ultrasonic echography. Embryonic losses were observed between Days 25 and 65 in 29.2% (7) Holstein and 31.8% (7) Japanese Black cows. The twin pregnancy rate in Holstein and Japanese Black cows decreased with time; 60.0% (9 of 15) vs. 37.5% (6 of 16) at Day 25; 53.8% (7 of 13) vs. 28.6% (4 of 14) at Day 45 and 41.7% (5 of 12) vs. 15.4% (2 of 13) at Day 65. At calving, Holstein cows produced five sets of twins and seven single calves, and Japanese Black cows two sets of twins and 11 single calves. The twinning rate in Holstein cows was higher (P < 0.05) than that in Japanese Black cows, 41.7% (5 of 12) vs. 15.4% (2 of 13). The calf birth weight in Holsteins was heavier (P < 0.05) than that in Japanese Black dams (24.5 kg, 33.6 kg vs. 19.3 kg, 25.5 kg for twin and single calves). The placental weight in Holstein dams calving twins was heavier than that in Holstein dams calving a single calf or in Japanese Black dams calving either twins or a single calf (6.6 kg vs. 3.5 kg, 4.6 kg or 2.8 kg). The number of placentome in Holstein dams calving twins was also higher (P < 0.05) than that in Holstein dams calving a single calf or Japanese Black dams (103.5 vs. 41.8, 67.9, 33.0). The number of placentome was approximately double in dams calving twins than that of dams calving a single calf.     

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.