Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Rederivation of Transgenic and Gene-Targeted Mice by Embryo Transfer

Research on genetically engineered mice provides insights into the etiology, therapy, and genetic basis of human diseases. An important variable that affects the results of mouse studies is the health status of the animals. Pathogen burdens may confound observations and obscure underlying mechanisms. Mouse resource centers frequently rederive infected mouse strains. We review our experience on the use of a well-established technique, embryo transfer to rederive infected mouse strains. The following mouse pathogens were eliminated by embryo transfer: Mouse Parvovirus, Mouse Hepatitis Virus, Mouse Rotavirus, Mouse Encephalomyelitis Virus, Mouse Adenovirus, Helicobacter species, endoparasites, and ectoparasites. We rederived transgenic mouse lines, gene-targeted mouse lines, and lines with spontaneous mutations. In the majority of strains, fertilized eggs for embryo transfer were obtained by mating superovulated egg donors with males of the desired genotype. A total of 309 embryo transfers were performed to rederive 96 mouse strains. The pregnancy rate was 76%; 1996 pups were born, of which 43% carried the desired genotype. We performed 44 additional embryo transfers to rederive 15 other strains. The pregnancy rate was lower (45%) and none of the 135 pups carried the desired genotype. Although we successfully eliminated the pathogens in all transfers, we were unable to obtain pups with the desired genotype in 15 of 111 mouse lines. Multiple factors affect the efficiency of rederivation by embryo transfer. They include the response to superovulation by embryo donors, the number and age of stud males, the yield of fertilized eggs, the number of embryo transfers, and genotyping.     

Risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes

The aim of this study was to estimate the risk of mouse hepatitis virus (MHV) transmission by the in vitro fertilization and embryo transfer (IVF-ET) procedure. In addition, resistance to infection of zona-intact and laser-microdissected oocytes was compared. For this purpose, infectious mouse hepatitis virus, a common viral pathogen in mouse facilities, was used. Oocytes having an intact or laser-microdissected zona pellucida were incubated for fertilization in media containing MHV-A59 and resulting embryos were transferred to the oviduct of specific pathogen-free (SPF) Swiss recipients. The oocytes were divided into three experimental groups: 1) zona-intact oocytes continuously exposed to MHV in fertilization (HTF), culture (KSOM), and embryo transfer (M2) media; 2) zona-intact oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2; and 3) laser-microdissected oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2. Respective serum samples of embryo recipients and their offspring were tested for MHV antibodies using ELISA. In experiment 1, 10 out of 14 embryo recipients seroconverted to MHV and only their offspring (8 of 19) received maternal antibodies. In experiments 2 and 3, MHV antibodies were detected neither in the recipients nor in the offspring. These results indicate, for the first time, that even if the zona pellucida is partially disrupted by laser microdissection, the transmission of MHV-A59 can be avoided by correctly performed washing steps in the IVF-ET procedure.     

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.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

两种玻璃化胚胎冷冻方法对不同品系小鼠胚胎冷冻效果比较

目的探讨EFS和DAP两种玻璃化冷冻方法对不同品系小鼠胚胎冷冻的效果。方法6个品系小鼠（KM、ICR、BALB／c、C57BL／6J、OB／OB、LAP／~TAOF59）的2-cell胚胎分别用EFS和DAP两种玻璃化冷冻方法进行冷冻和复苏,比较两种冷冻方法的胚胎复苏率和着床率。结果6个品系小鼠冷冻胚胎EFS方法的平均复苏率为69．97％（47．9％～83．6％）,DAP方法的平均复苏率47．23％（26．3％-76．7％）,EFS方法明显优于DAP方法。其中KM、ICR和BALB／c小鼠EFS方法的冷冻复苏率显著高于DAP方法（P〈0．01）;冻融胚胎移植后EFS方法的平均着床率27．23％（1．75％一45．0％）,DAP方法的平均着床率31．43％（7．0％一46．3％）。除KM、ICR小鼠外,其他4个品系小鼠的着床率DAP方法高于EFS方法。结论KM和ICR远交群小鼠胚胎适合用EFS方法冷冻保存;C57BL／6J、OB／OB、LAP／aTAOF59三个品系小鼠DAP方法优于EFS方法,但差异不大;BALB／c小鼠两种玻璃化冷冻方法的冻融胚胎着床率均较低,需进一步研究。     

Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs)

Superovulation, in vitro fertilization, embryo cryopreservation, and embryo transfer are assisted reproductive technologies (ARTs) widely used in laboratory mice. Inbred strains of mice have inherent genetic differences that cause them to respond differently to these technologies. Knowing how common inbred strains will perform when used for ARTs will ensure the most efficient use of mice, time, and resources. In this study, we characterized the ability of 10 inbred strains: 129S1/SvImJ, A/J, BALB/cJ, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, NOD/LtJ, and SJL/J to superovulate, fertilize in vitro, and produce live pups subsequent to embryo transfer. Three-week-old female mice were superovulated using eCG (5.0 IU) and hCG (5.0 IU). The resulting oocytes were fertilized in vitro in human tubal fluid medium with spermatozoa of the same strain. The following day, two-cell embryos were either transferred into pseudopregnant recipient females or cryopreserved. The cryopreserved embryos were later thawed and transferred into pseudopregnant recipient females. Differences in response to superovulation, fertilization, and number of live born produced after embryo transfer were observed between strains, substantiating the influence of genetic variability on ARTs. The response to the superovulation treatment varied among strains and ranged from 5+/-1(A/J) to 40+/-3 (129S1/SvImJ) normal oocytes per female. The average proportion of oocytes that fertilized ranged among strains from 24% (129S1/SvImJ) to 93% (DBA/2J and A/J). The average proportion of two-cell embryos that were transferred into recipient females and subsequently developed into live pups varied from 5% (A/J) to 53% (C57BL/6J) for fresh embryos and from 18% (BALB/cByJ) to 45% (129S1/SvImJ) for thawed embryos.     

Transfer of vitrified blastocysts from one or two superovulated Large White Hyperprolific donors to Meishan recipients: reproductive parameters at Day 30 of pregnancy

The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Intact blastocysts were collected from superovulated Large White Hyperprolific gilts (n = 24) on Days 5-5.5 after artificial insemination. Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as described by Berthelot et al. [Cryobiology 41(2000) 116]. To evaluate in vitro development, 37 vitrified/warmed blastocysts were cultured, non-vitrified embryos (n = 48) were used as controls. Embryo transfers were conducted in asynchronous (-24 h) Meishan gilts (n = 20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (Group 1) and the other 10 transfers were performed with mixed embryos from two donors (Group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered at Day 30 after transfer. In vitro survival rate of the vitrified/warmed blastocysts was lower (P < 0.01) than that from control embryos (73.0% versus 93.7%). The pregnancy rate for Group 1 (70%) was not different (P > 0.05) than that from Group 2 (90%). No significant differences were detected between Groups 1 and 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P < 0.05) when transfers were performed with embryos from two donors (19.5% versus 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts.     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Viability of blastocysts produced by aggregation of two half-embryos in the mouse

Although methods for production of chimeras from early cleavage stages have been well established, little research has been directed toward production of genetically identical chimeric offspring. This study was designed to examine survival of blastocysts produced by aggregation of two halved eight-cell stage embryos from two different mouse strains. Four blastomeres of an eight-cell embryo from a pigmented strain were aggregated with four blastomeres of an eight-cell embryo from a nonpigmented strain. Aggregates were cultured for 48 h and transferred as blastocysts to synchronized recipients of three treatment groups. Viability was determined by examining the number of offspring produced relative to the number of blastocysts transferred. Thirty-nine pups were born from 375 transferred blastocysts (10%), with 16 pups displaying coat-color chimerism. Both nonmanipulated eight-cell embryos cultured for 48 h (P < 0.05) and chimeric blastocysts (P < 0.001) displayed lower embryo survival after transfer to recipients than noncultured, nonmanipulated blastocysts used as controls. Viability of chimeric blastocysts was also lower than that of nonmanipulated embryos cultured for the same period and transferred to the same recipients (P < 0.001). Although posttransfer survival of chimeric blastocysts was low, the birth of morphologically normal offspring demonstrated that production of chimeras from half embryos was compatible with survival. Improvements in this procedure may be useful for production of tenetically identical chimeras from outbred populations, such as those commonly found in domestic livestock species.     

Acquisition of endogenous ecotropic MuLV can occur before the late one-cell stage in the genital tract of SWR/J-RF/J hybrid females

Endogenous ecotropic MuLV proviral loci are acquired by the progeny of some [SWR/J x (SWR/J x RJ/J)F1] N2 hybrid females obtained by two successive backcrosses of RF/J mice onto the SWR/J background. This results most likely from an infection of early embryos or oocytes by MuLV particles originating from maternal tissues. However, the time and site of infection are not yet known. Using oviductal transfers of embryos at the one-cell stage, we show here that three of 88 N3 embryos from [SWR/J x (SWR/J x RF/J)F1] N2 hybrid females transferred to virus-free foster mothers harbored new proviral integrations, whereas none of 61 SWR/J embryos transferred to [SWR/J x (SWR/J x RF/J)F1] N2 hybrid females had acquired any proviruses. These data support the infection of oocyte and/or early one-cell embryo as the initial event leading to new proviral insertions.     

An optimal embryo transfer condition for the effective production of DBA/2J mice.

The DBA/2J mouse strain is a standard laboratory strain that is widely used for biomedical research. This strain, however, suffers from poor reproductive performance. In addition, the conditions for reliable embryo transfer (ET) of this strain have not been elucidated. The intention of this study was to determine the optimal number of embryos for transfer that allow the effective production of DBA/2J offspring. In the experiment, 7 to 15 embryos per oviduct were transferred into pseudopregnant ICR females. A relatively high success rate for pup production was observed when a large number of DBA/2J embryos (30 embryos per female) were transferred. This result shows that the ET efficiency of the DBA/2J strain can be improved by increasing the number of transferred embryos.     

Donor and recipient genotype and heterosis effects on survival and prenatal growth of transferred mouse embryos

Reciprocal embryo transfers amongst two inbred strains (C3HeB/FeJ and SWR/J) and their F1 cross (C3SWF1) were used to examine donor and recipient genotype and heterosis effects on survival and prenatal growth of mouse embryos. Among inbred strains, significant recipient genotype effects were detected for both embryo survival (P less than 0.01) and prenatal growth (P less than 0.05), while no donor genotype effects were observed. The recipient effect on overall embryo survival was due to a higher proportion of C3H recipients maintaining pregnancy to term than SWR recipients (P less than 0.01), rather than survival within litters. Irrespective of their own genotype, embryos developing in C3H uteri achieved larger body weights (P less than 0.01) and longer tail lengths (P less than 0.05) at birth than did embryos developing in SWR uteri. Recipient heterosis was not significant, while donor heterosis was significant for prenatal growth traits (P less than 0.001).     

Long-term cryopreservation of mouse sperm

The objective was to determine if mouse sperm can maintain their fertilizing ability after being frozen for >10 y and whether the offspring derived from these sperm had normal fertilizing ability and phenotype. We cryopreserved sperm from six strains of mice (C57BL/6J, DBA/2N, BALB/cA, C3H/HeJ, B6D2F1 and B6C3F1) in a solution containing 18% (w/v) raffinose and 3% (w/v) skim milk, and preserved them in liquid nitrogen for >10 y. To assess the normality and fertilizing ability of these sperms, they were thawed and used for in vitro fertilization of oocytes of the same strains. Fertilization rates for C57BL/6J, DBA/2N, BALB/cA, C3H/HeJ, B6D2F1 and B6C3F1 were 66.4, 92.3, 72.8, 32.9, 60.3 and 53.7%, respectively. Furthermore, 38.3, 15.0, 43.3, 26.1, 38.3 and 16.7% of the embryos transferred to pseudopregnant females developed and produced live offspring that had normal phenotype and fertility.     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius

Dobson C., Brindley P. J. and Sitepu P. 1982. Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius. International Journal for Parasitology12: 567–572. Different strains of serum donor mice showed variations in innate immunity to primary infections with Nematospiroides dubius. Different levels of anti-N, dubius antibodies were detected in sera from these mouse strains; there was no correlation between antibody titre and numbers of worms recovered. Serum from donor wild and six laboratory strains of mice protected female Quackenbush (Q) recipients against N. dubius infections; donor mouse strain influenced the degree of protection conferred and donor serum antibody titre related to the degree of stunting of worm growth in recipient mice. Five laboratory strains of mice developed different levels of protective immunity following multiple experimental infections with N. dubius. Antibody titres in these mice were strongly correlated with the percentage protection observed after 1–4 infections: Q and CBA mice produced more anti-N. dubius antibody and were better protected than DBA/2, BALB/c and C3H mice. However BALB/c, C3H and CBA mice attained similar anti-N. dubius antibody titres after a single infection with N. dubius but serum from BALB/c gave better protection when transferred to female Q recipients than that from the other two strains. This suggested qualitative differences in the protective antibodies in sera between mouse strains. Five mouse strains were passively immunized with a uniform dose of serum from female Q donors: DBA/2 female recipients showed the least, BALB/c and C3H females were intermediate, and Q and CBA female mice attained the greatest level of passive protection against N. dubius. A close positive correlation existed between the degree of actively acquired and the level of passively acquired protection between the five strains of mice.     

Transmission of mouse minute virus (MMV) but not mouse hepatitis virus (MHV) following embryo transfer with experimentally exposed in vivo-derived embryos

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10(5)/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10(7) mean tissue culture infective dose (TCID(50))/ml of MHV-A59 and 10(2) TCID(50)/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.     

Phenotypic analysis of C57BL/6J and FVB/NJ mice generated using evaporatively dried spermatozoa

Combination of evaporative drying and frozen storage at -80 degrees C has been used successfully to preserve hybrid B6D2F1 mouse spermatozoa. To determine whether this method can be applied equally well to inbred mice, spermatozoa of C57BL/6J and FVB/ NJ mice were evaporatively dried and stored for 1 mo at -80 degrees C before being used for intracytoplasmic sperm injection (ICSI) to produce live offspring. After weaning, 1 male and 1 female mouse from each litter were randomly selected at 8 wk of age for natural mating to produce live offspring. Results showed that spermatozoa from both inbred strains that had been evaporatively dried and subsequently stored at -80 degrees C could be used successfully to derive live, healthy, and reproductively sound offspring by ICSI. No significant differences were found in embryo transfer rate (number of pups born/number of embryos transferred), litter size, weaning rate, body weight, number of pathologic lesions, and amount of contamination by pathogens of mice produced by ICSI using evaporatively dried spermatozoa compared with mice produced by natural mating or by ICSI using fresh (that is, nonpreserved) spermatozoa. Progeny produced by mating mice generated from ICSI using evaporatively dried spermatozoa were normal. Therefore, spermatozoa from inbred mouse strains C57BL/6J and FVB/NJ can be preserved successfully after evaporative drying and frozen storage at -80 degrees C.     

Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats

We investigated the capability of repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, to expedite the production of transgenic progeny from transgenic founder dairy goat does. In addition, we compared embryo yields, number of embryos transferred per recipient, pregnancy rates, and offspring born during both the traditional (September-December) and non-traditional (January-May) breeding seasons. Although there were no significant differences, there were numerically more transferable embryos recovered per flush (3.5+/-0.9 vs. 2.4+/-0.9 embryos; mean+/-S.E.M.) and increases in both the proportion of recipients that were pregnant (83 vs. 69% pregnant) and offspring born from total embryos transferred (67 vs. 53% offspring) during the traditional versus the non-traditional breeding season. The transfer of one, two or three embryos did not significantly affect the proportion of pregnant recipients during either season. However, there was a difference (P<0.05) in the proportion of offspring produced for one versus two embryo transfers (89 vs. 44% offspring, respectively) during the non-traditional breeding season. Overall, 14 transgenic offspring were produced from 54 total offspring born, and the kidding interval was reduced to <3 months for six of the seven transgenic does. In summary, repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, expedited the production of progeny from transgenic founder does.     

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.