Factors affecting success of embryo collection and transfer in a transgenic goat program

During a goat transgenic program that took place in Israel from July 1995 to February 1996, Saanen (n = 343) and Nubian x Damascus (n = 378) crossbred goats of mixed ages were used as donors (n = 433) and recipients (n = 288). The effects of season, age, number of surgical procedures, previous hormonal treatments and ovulation rate on the number of microinjectable embryos collected were studied. Likewise, the effects of these parameters on the pregnancy rate as well as the number of embryos transplanted, endogenous progesterone concentrations and exogenous progesterone supplementation were studied in recipient does. Following superovulation with ovine follicle stimulating hormone, 85% of the does responded with 13.6 +/- 5.7 (mean +/- S D) ovulations/doe. Age, month and number of previous hormonal treatments significantly affected the ovulation rate. The average recovery rate was 70%, and it was affected only by the ovulation rate. Pronuclei were visualized in about 30% of the flushed embryos (including unfertilized ova), and those were microinjected with human serum albumin gene construct. About 68% of the injected embryos underwent at least one division during an overnight incubation, and those embryos were transferred, giving about 2.0 transferred embryos per ovulated donor. Of the recipients, 86% responded following synchronization with 3.1 +/- 1.6 (mean +/- S D) ovulations per doe. Breed and month had a significant effect on the ovulation rate. Two or three microinjected embryos were transferred to each recipient, resulting in more than a 40% pregnancy rate during September to November. Lower pregnancy rates were obtained before and after that period. By monitoring plasma progesterone concentrations in the recipients it was found that progesterone concentration was correlated with the ovulation rate. However, the pregnancy rate was not affected by progesterone concentration. During January and February, 30 to 50% of the recipients failed to develop functional corpora lutea (CL) following embryo transfer, which explained the lower pregnancy rate in those months. Of the 86 kids born 4 were transgenic.     

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.     

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus = Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2alpha treatment on Days 6 and 7. However, the proportion of cows with > 2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with > 2 ovulations (31 vs 20%), ovulation rate. (26.0 +/- 6.3 vs 49.6 +/- 25.8), production of ova/embryos (13.3 +/- 3.2 vs 14.4 +/- 3.4), or the number of transferable embryos (8.0 +/- 3.6 vs 5.4 +/- 1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0 +/- 2.0 mm) was smaller (P<0.05) than at midcyle (16.8 +/- 1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.     

Offspring resulting from transfer of cryopreserved embryos in camel (Camelus dromedarius)

The dromedary embryos, collected at hatched blastocyst stage, survived freezing and thawing in the presence of a high concentration of ethylene glycol (7.0 mol/L) with sucrose (0.5 mol/L) and direct plunging in liquid nitrogen. The rate of survival, as judged by the morphological appearance of the embryos after thawing, was high (92%). The transfer of frozen-thawed embryos into the recipients during the breeding (n=20) and non-breeding season (n=25) resulted in two and one pregnancy, respectively. One of the two pregnant recipients, with embryos transferred during the breeding season, delivered a normal healthy male calf at term. To our knowledge, this offspring is the first camelid produced following transfer of a frozen-thawed embryo.     

DNA preparation method can influence outcome of transgenic animal experiments

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

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.     

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.     

Risk evaluation for bovine embryo transfer services using computer simulation and economic decision theory

A mathematical model was used to evaluate the dynamics and risks in bovine embryo transfer. Variables included embryo collection, fertilization, and transfer rates, plus overall pregnancy rates. Decision analysis was applied to three sets of 500 simulated flushes to test three strategies: 1) nonsuperovulation, 2) superovulation using follicle stimulating hormone (FSH) and 3) superovulation using pregnant mare serum gonadotropin (PMSG). Model validation involved comparing model performance against standards derived from 39 published studies. The model's repeatability was +/- 0.10 against standards in 93% of cases, and never exceeded +/- 0.13 in all 1500 simulations. Model outcomes were accurate to +/- 3% using average results. No pregnancies occurred in 22% of nonsuperovulated donors, 8% of FSH superovulated donors, and 6% of PMSG superovulated donors. PMSG treatment averaged more pregnancies per flush than FSH treatment (4.4 vs 3.9) but showed greater variation in response (64 vs 51%). Decision analysis suggests that a PMSG-induced flush would net $105 more than an FSH-induced flush, and that either superovulation strategy would yield approximately 10 times the net income of a nonsuperovulated flush. Pricing by response (PMSG) or by transfer (FSH) is optimal for the provider of embryo transfer services.     

Normal reproductive capacity of heifers that originated from in vitro fertilized embryos cultured with an antiviral compound

Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers.     

Effect of dietary protein on embryo recovery rate and quality in superovulated heifers

For almost 3 decades, superovulation and embryo transfer have been used in cattle breeding to increase the number of offspring from genetically superior female animals. Several factors including nutrition affect the number of transferable embryos recovered. We compared the effects of two different dietary protein levels easily achieved in practical conditions on embryo number and quality in superovulated heifers. Finnish Ayrshire heifers (n = 37) were allocated to isoenergic diets containing either 14% (D14) or 18% (D18) crude protein (CP). Estruses were synchronized, and the heifers were subsequently superovulated and inseminated using a standard FSH-protocol. Embryos were collected 7 days after inseminations (71-72 days after the beginning of the treatment period) by uterine flushing. The number of corpora lutea, and the number and quality of embryos were determined. Protein feeding did not affect superovulatory response, the number of embryos or the number of transferable embryos recovered. Proportionally more poor-quality embryos were found in group D14 than in group D18 (20.2% versus 13.2%, respectively, P = 0.053). It is concluded that a long-term moderate increase in the content of crude protein fed to energy-adequate heifers does not seem to affect superovulatory response and the number of embryos recovered, but it may be advantageous to the quality of embryos.     

Field evaluation of juvenile in vitro embryo transfer (JIVET) in sheep

The practicality of using juvenile in vitro embryo transfer (JIVET) on a field scale in China was evaluated in each of three seasons (summer, autumn and winter) from 2006 to 2007. A total of 102 donor Merino lambs (18 summer, 69 autumn and 15 winter) aged 4-8 weeks were stimulated with 4 x 40 mg FSH administered at 12h intervals plus 400 IU PMSG given at the time of the first FSH treatment. Overall, 89.2% (91/102) of the lambs exhibited follicle development and 79.1+/-65.5 (mean+/-S.D.) cumulus-oocyte complexes were recovered per donor lamb. Compared with the groups of summer (84.9+/-55.3) and autumn (83.6+/-70.8) lambs, the number of recovered cumulus-oocyte complexes was significantly decreased in winter (51.4+/-43.7; p<0.05). After recovery, the cumulus-oocyte complexes were matured and fertilized in vitro using frozen-thawed semen and culture in synthetic oviduct fluid medium to the 2-4-c stage of development, when they were transferred surgically in groups of 3-8 (5.33+/-1.47) to the ipsilateral uterine horn of a total of 603 synchronized recipients. The overall mean proportion of cumulus-oocyte complexes developing to 2-c embryos was 61.4% (4308/7013) and differed significantly between seasons (summer 38.5%, autumn 66.1%, winter 74.6%; p<0.01). Pregnancy rate assessed by ultrasound examination approximately 60 days after embryo transfer was 54.4% (328/603) overall, and 36.7% (221/603) of the recipients maintained their pregnancy to full-term, producing an average 1.49 (330/221) offspring, of which 1.21 (267/221) were viable and healthy lambs, per pregnant recipient. Pregnancy rate at day 60 was affected by season (summer 40.5%, autumn 56.7%, winter 55.7%; p<0.05), but did not differ significantly between seasons at full-term (summer 34.2%, autumn 38.9%, winter 30.4%; p>0.05). Based on the number of donors stimulated, the total number of offspring and viable progeny produced per donor lamb in autumn (5.81 and 4.87) was significantly (p<0.01) higher than that of summer (2.79 and 1.94) and winter (4.24 and 3.31). This study showed that each donor lamb after stimulation produced an average of 48.6 transferable embryos that resulted in 4.04 viable and healthy progeny. These results indicate that JIVET is a cost-effective method of multiplying desirable sheep genotypes in China.     

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.     

Transfer of superovulated sheep embryos obtained with different FSH-P

Embryo transfer is one way of accelerating genetic improvement in sheep. One of the main obstacles has been the production of good-quality embryos. The use of progestagens and the stimulation of ovulation with follicle stimulating hormone pituitary extract (FSH-P) has permitted the superovulation of donor and recipient ewes and the synchronization of their cycles. The injection of 16 mg FSH-P at the end of progestin treatment gave means of 9 +/- 1.5, 12 +/- 1.5, and 19.5 +/- 2.6 corpora lutea per ewes in the Préalpes, Lacaune, and Romanov x Préalpes breeds respectively (this last breed is particularly prolific). Twenty Préalpes donor ewes produced 133 embryos that were recovered surgically at Day 6 of gestation; of these, 99 morulae were transferable. Forty-five morulae transferred surgically into 24 Préalpes recipient ewes yielded 16 pregnant ewes and 27 lambs (1.7 per ewe). Twenty-two Lacaune ewes yielded 204 embryos, of which 152 morulae were transferable. Of 76 recipients, 58 became pregnant and gave birth to 97 lambs (1.7 per ewe). During anoestrus, the mean ovulation rate decreased from 11.2 to 8.4; 40.6% of the embryos recovered were of transferable quality versus 74.5% during the normal breeding season. An improved superovulation technique, based on the use of FSH-P with a known follicle stimulating hormone to luteinizing hormonal (FSH/LH) ratio, provided us with good-quality embryos. This treatment must be adapted to the season.     

Donor category and seasonal climate associated with embryo production and survival in multiple ovulation and embryo transfer programs in Holstein cattle

The present study investigated the effect of Holstein donor category (cows vs. heifers) and climate variation (hot vs. cooler season) on the efficiency of in vivo embryo production programs as well as embryo survival after transferred to Holstein recipient cows. A total of 1562 multiple ovulation (MO) procedures (cows: n = 609, and heifers: n = 953) and 4076 embryo transfers (ETs) performed in two dairy herds were evaluated. Donor cows had greater number of CLs (10.6 ± 0.6 vs. 7.5 ± 0.4; P < 0.0001) and ova/embryos recovered (7.6 ± 0.6 vs. 4.6 ± 0.4; P < 0.0001) compared with donor heifers. However, fertilization rate (47.9 vs. 82.4%; P < 0.0001) and proportion of transferable embryos (31.5 vs. 67.4%; P < 0.0001) were lower in donor cows than heifers, respectively. Regardless of donor category, the proportion of freezable embryos was less (P < 0.001) during hot season than in cooler season (21.4 vs. 32.8%). However, greater decline in the proportion of freezable embryos during the hot season was observed in cows (21.7 vs. 10.7%) compared with heifers (46.2 vs. 38.1%; P = 0.01). In contrast, the season on which the embryo was produced (hot or cool) did not affect pregnancy rate on Day 31 (30.5 vs. 31.7%; P = 0.45) and 45 (25.3 vs. 25.1%; P = 0.64) of pregnancy. Regardless of the season in which the embryos were produced, embryonic survival after transferring embryos retrieved from donor cows was greater on Days 31 (36.0 vs. 30.7%; P = 0.001) and 45 (28.3 vs. 23.1%; P = 0.001) of pregnancy when compared with embryos from donor heifers. In conclusion, MO embryo production efficiency decreased during the hot seasons both in cows and heifers; however, the decline was more pronounced in donor cows. Regardless of the embryo source, similar pregnancy rate was observed in the recipient that received embryos produced during the hot and cooler seasons. Curiously, embryos originating from donor cows had higher embryonic survival when transferred to recipient cows than embryos originating from heifers.     

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

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

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.     

Factors influencing embryo transfer success in alpacas—A retrospective study

Embryo transfer offers great advantages to South American camelid farmers to reach their breeding goals but the technology still plays a relatively minor role in comparison to other domestic farm animals like cattle. The aim of the present study was to analyse a data set of 5547 single or multiple ovulation embryo transfers performed in commercial alpaca farms in Australia to determine the factors that influence number and quality of embryos produced, embryo transfer success (percentage of crias born) and gestation length following transfer. Logistic binary regression identified the variables day of flushing after mating, embryo diameter, embryo quality, day of transfer after GnRH, and the age of the recipient to have significant impact on the outcome measure embryo transfer success. Transfer of smaller embryos or lower quality embryos resulted in decreased transfer success rates. Optimal days for obtaining embryos from donors were Days 8 and 9 after mating, optimal days for transfer into recipients were Days 7 and 8 after GnRH treatment. Age (>15 years) and body condition of recipients <2 also lowered transfer success rates, while the summer heat had no adverse impact. However, season did influence gestation length, while cria gender did not. In conclusion, results from the analysis of this very large dataset can underpin new recommendations to improve embryo transfer success in alpacas.     

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.     

Transuterine embryo migration in recipient cattle

Transuterine migration of bovine embryos following fertilization in vivo is apparently rare, but little is known about migration following embryo transfer. We studied heifers receiving either 1 or 2 in vitro produced embryos to determine 1) the incidence of transuterine migration, 2) the timing of migration and 3) the random or systematic occurrence of the event. In 4 experiments, 436 heifers received embryos and 218 of these were pregnant at necroscopy on either Day 14, Day 18, Day 26 or Day 60 of pregnancy. Overall, 43/218 (20%) of the heifers had embryos that had migrated. The frequency of migration was higher in twin (30/68) than in single (13/150) embryo transfers of pregnant recipients (44 vs 9%; P<0.001), and in contralateral (9/15) than in ipsilateral (33/170) transfers (60 vs 19%; P<0.001). Among the heifers that received embryos by ipsilateral transfer, the migration rate was similar to that in heifers pregnant with a singleton after the transfer of either 1 (2/48) or 2 (4/60) embryos (4 vs 7%, NS). The migration rate was highest at Day 26 (12/37) in heifers receiving twin embryos by ipsilateral transfer but was similar at all other stages of pregnancy (15/111, 32 vs 14%; P<0.01). Migration was first observed by Day 14, and it appears that either further migration occurred over the next 12 d or that migration was associated with a higher survival rate from Day 14 to Day 26. The low migration rate evident at Day 60 suggests that migration by Day 26 was associated with increased embryo or fetal death by Day 60. The data suggest that embryo migration is probably independent for each of a pair of surviving embryos. We conclude that in cattle embryo migration is embryo-dependent, but this capability is dormant unless more than 1 embryo is present in a uterine horn or the embryos are transferred to the contralateral uterine horn. The relationship between migration and embryo survival remains unclear.