Failure to transmit scrapie infection by transferring preimplantation embryos from naturally infected donor sheep

The objective of the study was to examine whether or not the preimplantation embryo can act as a carrier of classic scrapie infection. The study was carried out on quarantined premises with sheep of highly susceptible scrapie genotypes. Uninfected embryos, collected from New Zealand–derived Suffolk ewes, were surgically transferred into recipient ewes that were also of New Zealand origin. Seventeen negative control lambs were born on the study premises from these embryo transfers. Thirty-nine experimental lambs were from embryos collected from naturally infected donor ewes. The experimental lambs were also born on the study premises after their surgical transfer into recipient ewes of New Zealand origin. These embryos had been collected from donor ewes in a scrapie-infected flock where the ewes were clinically sick with scrapie or developed clinical scrapie after embryo collection. All lambs were confirmed as scrapie susceptible of the ARQ/ARQ genotype. Twenty-eight experimental animals survived to the end point of the study at 5 yr of age with a mean survival of 1579 d. In the negative control group, 12 of 17 sheep survived to 5 yr of age with a mean survival of 1508 d. Postmortem examinations were carried out on all animals derived by embryo transfer, and in none was histologic or immunohistochemical evidence of scrapie found. In contrast, in the originating flock the majority of scrapie cases occurred in ARQ/ARQ genotyped animals where a 56% mortality from scrapie had been recorded in animals of this genotype. Thus, the study provides no evidence for transmission of scrapie and reinforces published evidence that vertical transmission of scrapie may be circumvented by embryo transfer procedures.     

Polymorphic distribution of the ovine prion protein (PrP) gene in scrapie-infected sheep flocks in which embryo transfer was used to circumvent the transmissions of scrapie

The genetic sequence of the ovine prion protein (PrP) gene between codons 102 and 175 with emphasis on ovine PrP gene codons 136 and 171 was determined, and the polymorphic distribution of the ovine PrP gene in the scrapie-exposed Suffolk embryo donors and offspring from these donors that were transferred to scrapie-free recipient ewes was investigated in this study. The most common genotype was AA(136)QQ(171) (70% and 63% in the donor and offspring flocks, respectively), which is considered a high risk genotype in US Suffolk sheep. Although embryos were collected from scrapie-positive donors and many embryos had the high risk genotype, no scrapie occurred in the resulting offspring. Based upon the results of this study, we conclude that vertical transmission of scrapie can be circumvented using embryo transfer procedures even when the offspring have the high risk genotype.     

Prp-c and Prp-Sc at the fetal-maternal interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.     

Evidence in Sheep for Pre-Natal Transmission of Scrapie to Lambs from Infected Mothers

Natural scrapie transmission from infected ewes to their lambs is thought to occur by the oral route around the time of birth. However the hypothesis that scrapie transmission can also occur before birth (in utero) is not currently favoured by most researchers. As scrapie is an opportunistic infection with multiple infection routes likely to be functional in sheep, definitive evidence for or against transmission from ewe to her developing fetus has been difficult to achieve. In addition the very early literature on maternal transmission of scrapie in sheep was compromised by lack of knowledge of the role of the PRNP (prion protein) gene in control of susceptibility to scrapie. In this study we experimentally infected pregnant ewes of known PRNP genotype with a distinctive scrapie strain (SSBP/1) and looked for evidence of transmission of SSBP/1 to the offspring. The sheep were from the NPU Cheviot flock, which has endemic natural scrapie from which SSBP/1 can be differentiated on the basis of histology, genetics of disease incidence and strain typing bioassay in mice. We used embryo transfer techniques to allow sheep fetuses of scrapie-susceptible PRNP genotypes to develop in a range of scrapie-resistant and susceptible recipient mothers and challenged the recipients with SSBP/1. Scrapie clinical disease, caused by both natural scrapie and SSBP/1, occurred in the progeny but evidence (including mouse strain typing) of SSBP/1 infection was found only in lambs born to fully susceptible recipient mothers. Progeny were not protected from transmission of natural scrapie or SSBP/1 by washing of embryos to International Embryo Transfer Society standards or by caesarean derivation and complete separation from their birth mothers. Our results strongly suggest that pre-natal (in utero) transmission of scrapie may have occurred in these sheep.     

Factors influencing success of embryo transfer in sheep and goats

The results of embryo transfers from 130 donor Angora goats and 60 sheep of 3 breeds are presented, and the data analyzed to determine some of the sources of variation in success rate. Of all adult donor goats programmed, 94.9% yielded embryos suitable for transfer and 93.4% yielded offspring from the transfers. Donor ewes yielded percentages of 76.8 and 46.7, respectively. Fertilization failure and/or degeneration of embryos in donors prior to flushing accounted for the lower recoveries of viable embryos from sheep, the incidence of both being greater in donors with higher ovulation rates. High ovulation rate of donors also decreased percentage survival of sheep but not goat embryos after transfer. Stage of embryo development, site of transfer (oviduct vs. uterus) or number of embryos transferred (1 vs. 2) per recipient did not affect survival of sheep embryos following transfer to appropriately synchronized recipients. In goats, survival was significantly better with two than with one embryo transferred per recipient. Super-ovulation failure and poor fertilization limited the yield of embryos obtained from donor goats and sheep less than 1 year of age. These could be overcome to some extent by use of progestagen sponge rather than prostaglandin in the superovulation treatment regimen.     

Risks of transmitting ruminant spongiform encephalopathies (prion diseases) by semen and embryo transfer techniques

Early experiments suggested that scrapie transmission via sheep embryos was a possibility, and gave rise to much controversy. However, when account is taken of the complex genetic effects on ovine susceptibility to scrapie, and of the several different scrapie strains with different clinical and pathological effects, the overall conclusion now is that transmission of classical scrapie by embryo transfer is very unlikely if appropriate precautions are taken. Recent embryo transfer studies have confirmed this. Other studies in sheep have shown that from about the middle of pregnancy the placental trophoblast is liable to scrapie infection in genetically susceptible ewes if the fetus is also susceptible. Since the contrary is also true, use of resistant ewes as embryo recipients could add to the safety of the embryo transfer, at least for classical scrapie. There has been little recent research on scrapie transmission via semen in sheep, and, with hindsight, the early studies, though negative, were inadequate. There is scant information on scrapie transfer via goat semen or embryos, although one study did find that bovine spongiform encephalopathy (BSE) was not transmitted via goat embryos. In cattle it has been shown that, if appropriate precautions are taken, the risks of transmitting BSE via semen and in vivo-derived embryos are negligible, and this conclusion has gained worldwide acceptance. Research on TSE transmission via reproductive technologies in deer has not yet been done, but information on the pathogenesis and epidemiology of chronic wasting disease (CWD) of deer, and on transmission risks in other species, provides optimism that transmission of CWD via semen and embryos of deer is unlikely. The presence of TSE infectivity in blood and various other tissues of infected animals, particularly sheep, gives rise to concerns that certain biological products currently used in reproductive technologies, e.g. pituitary gonadotrophins for superovulation, and certain tissue and blood products used in semen and embryo transfer media, could carry TSE infectivity. Instruments such as laparoscopes used for insemination, and for collection and transfer of embryos, especially in small ruminants, are also a concern because effective decontamination can be very difficult.     

Laparoscopic embryo transfer in domestic sheep: A preliminary study

An effective, minor-invasive technique for embryo transfer in sheep was developed using a laparoscopic transabdominal approach. Twelve recipient ewes received embryos either by conventional laparotomy or by laparoscopy. The estrous cycle of recipient ewes was synchronized using a progestagen-impregnated vaginal pessary/pregnant mares' serum gonadotropin treatment regimen. Donor ewes were superovulated with follicle stimulating hormone or human menopausal gonadotropin, bred with a ram of one breed and laparoscopically inseminated $\text{in}$$\text{utero}$ with semen from a different sheep breed. Five to six days after estrus, embryos were transferred laparoscopically into the terminal one-half of the recipient's uterine horn ipsilateral to the ovary with prominent corpus luteum development. Pregnancy was diagnosed by transrectal ultrasonic procedures, and by direct laparoscopic examination of the uterus. Of six laparoscopic transfers, three resulted in single births; one of six laparotomy transfers resulted in a live birth. Breed appearances of the four lambs born indicated that two of the offspring resulted from laparoscopic artificial insemination of the donor ewe. The results demonstrated that laparoscopic transfer of embryos was a rapid and safe procedure, easily applied to an ovine embryo transfer program and with potential for similiar studies in other species.     

Transgenic sheep generated by lentiviral vectors: safety and integration analysis of surrogates and their offspring

The safety of HIV-1 based vectors was evaluated during the production of transgenic sheep. Vectors were introduced into the perivitelline space of in vivo derived one-cell sheep embryos by microinjection then transferred into the oviducts of recipient females. At 60–70 days of gestation, a portion of the recipients were euthanized and tissues collected from both surrogates and fetuses. Other ewes were allowed to carry lambs to term. Inadvertent transfer of vector from offspring to surrogates was evaluated in 330 blood and tissue samples collected from 57 ewes that served as embryo recipients. Excluding uterine contents, none of the samples tested positive for vector, indicating that that the vector did not cross the fetal maternal interface and infect surrogate ewes. Evaluating ewes, fetuses and lambs for replication competent lentivirus (RCL); 84 serum samples analyzed for HIV-1 capsid by ELISA and over 600 blood and tissue samples analyzed by quantitative PCR for the VSV-G envelopes revealed no evidence of RCL. Results of these experiments provide further evidence as to the safety of HIV-1 based vectors in animal and human applications.     

Donor and recipient ewe factors affecting in vitro development and post-transfer survival of cultured sheep embryos

Donor and recipient factors were assessed during development of embryos following superovulation, collection at the pronuclear and two-cell stage, culture in Synthetic Oviduct Fluid medium for 5 days and twin transfer into synchronised recipients to elucidate what factors affect embryo development and post-transfer survival. In particular, the administration of exogenous progesterone to recipients using an intravaginal CIDR^TM device immediately following embryo transfer was investigated.

From 138 embryos collected from 30 donor ewes, 75% (103) were of transferable quality following culture, of which 100 were transferred to 50 recipients. There was significant variation (P < 0.001) in embryo development to the blastocyst stage between different donor ewes, but this was not related to the donor ovulation rate. At ultrasound sonography (approximately Day 60 of pregnancy), 58% of recipients were pregnant and 42% embryos had survived. Donor ovulation rate was related to embryo survival (P < 0.05) after transfer; the survival rate of embryos from ewes with high ovulation rates was lower than that of embryos from ewes with low ovulation rates. Exogenous progesterone supplementation following transfer did not affect embryo survival, rate of embryo development or plasma progesterone levels. In general, the results from this study suggest that factors other than efficacy of embryo culture can affect the outcome of embryo survival following transfer and that, where possible, these factors should be considered and balanced in experimental designs.     

The transmission of bluetongue virus by embryo transfer in sheep

The susceptibility of sheep to intrauterine infection with bluetongue virus (BTV) was established by introducing 10(4) plaque-forming units of BTV type 10 into the uterine lumen of two seronegative ewes in a simulated embryo transfer operation. Both ewes became viraemic and underwent seroconversion to BTV. Embryos recovered from seronegative superovulated donor ewes were incubated in vitro for 8 h with BTV type 10. After incubation the embryos were thoroughly rinsed by repeated transfer to sterile culture medium, and 12 of these embryos were then transferred to the uterus or oviduct of seronegative, synchronized recipients. Viraemia and seroconversion were detected in nine recipient ewes. Embryos recovered from eight viraemic ewes were transferred to 15 seronegative, synchronized recipients. Viraemia and seroconversion were detected in 2 of the recipients, both of which also became pregnant. A lamb born to a ewe becoming infected at the time of embryo transfer was clinically normal, and no evidence of BTV infection was obtained at postmortem examination of the lamb after slaughter.     

Factors affecting the survival of sheep embryos after transfer within a MOET program

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher (P<0.05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage (P<0.05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived significantly (P<0.05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher (P<0.05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results confirm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep.     

影响杜泊羊冷冻胚胎移植成功率的因素

采用引进的杜泊羊冷冻胚胎,以云南当地绵羊为受体进行胚胎移植。同期发情处理了158只受体羊,同期发情率为82．91％;对102只进行了胚胎移植,实际移植率为77．86％,3个情期内移植妊娠率达74．5％;出生68只,产羔率达66．7％。分析表明,胚胎发育阶段及级别、卵巢黄体情况、以及胚胎移植技术熟练程度直接影响胚胎移植成功率;此外,受体羊的处理程序及移植后的饲养管理、移植时机的把握、移植季节以及胚胎冷冻及解冻方法也会影响杜泊羊移植妊娠率,进而影响产羔率。     

Survival of sheep x goat hybrid inner cell masses after injection into ovine embryos

Modified blastocyst injection techniques were used to inject immunosurgically isolated sheep x goat hybrid inner cell masses (ICM) into ovine blastocysts, with subsequent transfer of composite embryos to ovine recipients. Hybrid embryos were collected from does artificially inseminated with Barbados ram semen. A total of 13 live and 2 aborted offspring resulted from the 34 composite embryos transferred to recipient ewes (38% embryo survival). Of the 15 offspring, 4 exhibited phenotypic hybridism and 2 (13%) of these were determined to be hybrid mean value of -sheep chimeras by karyotype, serum protein and isoenzyme analyses, and fiber identification. Each of the 4 was produced by an injection procedure that involved damage of the ovine host ICM. One additional offspring was unusual in appearance, but the presence of hybrid cells was not proven. Similarly, caprine ICM were immunosurgically isolated and injected into ovine blastocysts that were then transferred to ovine recipients. Of the 13 composite embryos transferred, 12 offspring were produced (92% embryo survival). Eleven were overt goat mean value of -sheep chimeras and, of these, 7 were also blood chimeras. The hybrid ICM was shown to be capable of contributing to normal embryonic and fetal development after injection into an ovine blastocyst but may be less likely to be incorporated with the ovine host ICM than is the caprine ICM.     

Plasma progesterone profiles,ovulation rate,donor embryo yield and recipient embryo survival in native Saloia sheep in the fall and spring breeding seasons

The response to superovulatory (SOV) and estrus synchronization (ES) treatments and the fertility of donor (n=68) and recipient (n=118) Saloia ewes was evaluated in the fall and spring breeding seasons. The proportion of acyclic ewes at treatment time was significantly higher in the spring than in the fall (42.6% versus 4.0%, P<0.00001). Donors treated with eCG had a significantly higher mean number of follicles over 5mm in diameter in the ovaries at embryo recovery and a significantly lower mean efficiency of recovery than FSH-treated ewes. These negative effects were more pronounced in the fall than in the spring, which resulted in a significantly lower mean number of total and fertilized ova recovered from eCG-treated ewes, compared to FSH donors in the fall, but not in the spring. Season had no significant effect on the ovulation rate and plasma P4 concentrations of recipients treated with a progestagen plus eCG combination. Although the recipient lambing and embryo survival rates were higher in the fall than in the spring the differences were not significant. No significant differences were observed in the ovulation rate or P4 concentrations of recipients that lambed compared to those that did not lamb. These preliminary results show that, in Portugal, response of Saloia ewes to SOV or ES treatments and donor fertility following the SOV treatment were similar in the spring and the fall, which suggests that in the spring acyclic ewes are in moderate anestrus. The effect of season on fertility following embryo transfer should be confirmed in further studies involving a larger number of animals. The semilaparoscopic transfer method reported here allowed lambing and embryo survival rates higher (although not significantly) than a standard surgical approach.     

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.     

Improving delivery and offspring viability of in vitro-produced and cloned sheep embryos

Recently developed, assisted reproductive technologies (e.g., in vitro embryo production and nuclear transfer) have encountered perinatal morbidity/mortality of the offspring produced, which are likely to hinder the application of these techniques. Consequently we have sought to develop a system of hormonal stimulation that will ensure the delivery of offspring more prepared for extrauterine life. Here we examine deliveries outcome in sheep carrying in vitro-produced and nuclear transfer (NT) embryos in comparison to artificially inseminated and naturally mated control ewes. All groups (excluding NT, which received one treatment) were subjected to one of two hormonal treatments for induction of delivery, whereas the third part of each group was left without any treatment. The first (commonly used for naturally mated ewes) dexamethasone treatment did not solve a majority of parturition disturbances, and actually the number of deliveries necessitating assistance was reduced (P < 0.05) by this treatment in the control group. On the other hand, combined estradiol plus betamethasone stimulation (E + B) solved a majority of complications regarding delivery performance such as lack of the preparation of the mammary gland, low myometrial contractility, insufficient cervical ripening, and impaired maternal behavior. Moreover, substantial reduction of neonatal mortality was observed following the combined treatment. In conclusion, the E + B induction of delivery overcame the majority of physiological and behavioral intrapartum failures of sheep foster mothers and increased the survival of offspring, and thus can be recommended as a safe method for inducing delivery in foster mothers carrying in vitro-generated embryos.     

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.     

Nuclear transfer in the bovine embryo: a comparison of 5-day, 6-day, frozen-thawed, and nuclear transfer donor embryos

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.     

Melatonin treatment of embryo donor and recipient ewes during anestrus affects their endocrine status, but not ovulation rate, embryo survival or pregnancy

Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer.     

Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer

Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes ("therapeutic cloning").