The use of embryo transfer in the field for increased calf crops in beef and dairy cattle

Non-surgical transfer of one embryo to 63 previously synchronized inseminated recipients was performed on farms to induce twinning in beef and dairy cattle. All the transfers were done by technicians of A.I. centers. After calving, a twinning rate of 44.4% was achieved with no differences between beef Charolais heifers (45.4%), lactating Normande cows (42.8%) and dairy Friesian cows (45.4%). Compared with controls, the calving crop after a three-breeding-cycle period was significantly higher with recipients: 1.16 calf per treated recipient vs. 0.83 calf per control animal. It would seem that the possibility of obtaining about one-third more calves with embryo transfer could be of economical interest to increase meat production from cattle herds.     

Non-surgical embryo transfer in cattle

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

The use of nuclear transfer to produce transgenic pigs

Manipulation of the pig genome has the potential to improve pig production and offers powerful biomedical applications. Genetic manipulation of mammals has been possible for over two decades, but the technology available has proven both difficult and inefficient. The development of new techniques to enhance efficiency and overcome the complications of random insertion is of importance. Nuclear transfer combined with homologous recombination provides a possible solution: precise genetic modifications in the pig genome may be induced via homologous recombination, and viable offspring can be produced by nuclear transfer using cultured transfected cell lines. The technique is still ineffective, but it is believed to have immense potential. One area that would benefit from the technology is that of xenotransplantation: transgenic pigs are expected to be available as organ donors in the foreseeable future.     

Improving successful pregnancies after embryo transfer

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo.We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7-10 days after transfer.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.     

Plasma progesterone profiles and factors affecting embryo-fetal mortality following embryo transfer in dairy cattle

The relationship between plasma progesterone (P4) levels and embryo survival, and the value of P4 profiles for the selection of cattle embryo transfer recipients is still a matter of controversy. This study reports a comparison between lactating cows and heifers (n = 407) from a single dairy herd, after transfer of either fresh or frozen-thawed good quality embryos, of their ability to sustain embryo-fetal development to term. Plasma P4 concentrations on the day of estrus (Day 0 = D0), Day 4, Day 7 and on Day 21 were measured and related to embryo survival. Plasma P4 levels on Days 0, 4 and 7 were similar in recipients later found pregnant or open. Plasma P4 levels on Day 7 were significantly higher (P < 0.01) in heifers than in cows, but they were similar in pregnant and nonpregnant heifers and in pregnant and nonpregnant cows. Pregnancy rates for fresh and frozen-thawed embryos were higher in heifers than in cows, but the differences did not reach significance. However, the overall late embryonic mortality was significantly higher (P < 0.01) and the calving rate for frozen-thawed embryos was significantly lower (P < 0.05) in cows than in heifers. As expected, plasma P4 on Day 21 was significantly higher (P < 0.001) in pregnant than in nonpregnant recipients, but there was no difference between pregnant cows and pregnant heifers. Plasma P4 levels on Day 7 of recipients presumed pregnant on Day 21 and later found pregnant or nonpregnant were similar, but plasma P4 levels on Day 21 were significantly higher (P < 0.001) in pregnant than in nonpregnant recipients. The results of this study suggest that plasma P4 levels until the day of transfer, except for the rejection of recipients with abnormal luteal function, are of limited practical use for embryo transfer recipient selection. However, in lactating cows low plasma P4 values on Day 7 might negatively affect embryo survival, while in heifers this effect is not noticeable. Lactating cows are more prone to embryo loss than heifers, especially in the case of frozen-thawed embryos; this is associated with a lower competence of the corpus luteum at Day 7.     

Statistical models predicting embryo survival to term in cattle after embryo transfer

Embryo survival to term in recipient cattle is highly variable. We examined calving data in the published literature to determine whether a model of binomial independence or a model which includes an embryo (e) and recipient term (r), adequately explain observed embryo survival rates following attempts to induce twin calving using transfer of two embryos. To achieve this we examined 32 published papers which provided us with 47 sets of data concerning 4560 recipients with either 0, 1 or 2 calves born. In each set of data, the observed embryo survival rate to term (p) (number of calves born/number of embryos) was calculated and the expected number of recipients with either 0, 1 or 2 calves born was determined, assuming a binomial distribution. Parameters for the second model were estimated using maximum-likelihood procedures. The model of embryo independence was rejected in 85% of the sets of data, suggesting that factors other than the embryo are important sources of variation in embryo survival or loss. The proposed e and r model of embryo survival adequately describes the published data in recipients receiving either single or twin embryos. In general, only 50-70% of embryos and recipients are sufficiently competent to result in a calving. Variation among laboratories producing either in vitro or in vivo derived embryos was due to variation in recipient and not embryo competence. It is argued that e rather than observed embryo survival rate, and r rather than observed pregnancy rate, should be used to compare differences among embryo treatments and groups of recipients, respectively. Acceptance of this proposition should permit faster progress in identifying the biology of superior embryos and recipients, which is a prerequisite to improving embryo survival rate in cattle. Collectively, the published data are not consistent with a model of embryo independence, and that a model of embryo survival to term which recognises recipient as well as embryo contributions to embryo survival may be more appropriate in cattle.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Endocrine profiles of somatic nuclear transfer-derived pregnancies in dairy cattle

In cattle, several hormones and proteins are necessary for maintenance of a normal pregnancy that will result in a viable calf. Deviation from the normal cascade or expected profile of reproductive hormones and proteins may be associated with impairment of somatic nuclear transfer-derived pregnancies and the high rate of fetal loss. The objectives of this study were to characterize maternal plasma concentrations of pregnancy-specific protein B (PSPB), progesterone (P4), estrone sulphate (E₁S), and estradiol (E2) during the last two-thirds of pregnancy (cloned calves), and to determine associations with gestational abnormalities. Cows with cloned fetuses, produced by either commercial (N = 16) or zona-free (N = 4) cloning techniques, were compared with pregnant animals derived from traditional embryo transfer (N = 6) or AI (N = 6), at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0 = estrus). Fetal well-being was monitored with ultrasonography throughout gestation. At Day 80, progesterone concentration was lower (P < 0.0001) in nuclear transfer (NT) recipients than in control groups. Mean estrone sulphate concentrations did not vary significantly between NT and control groups. At Day 150, pregnancy-specific protein B concentrations were elevated (P < 0.002) in NT cows. Estradiol concentration was higher in NT recipients than control cows throughout the study period.     

Non-surgical embryo transfer in cattle embryo-recipient interactions

Results from 2286 non-surgical embryo transfers were analyzed to find relationships between embryo factors and recipient factors that affect pregnancy. Pregnancy rates from morulae (44%) and advanced morulae (53%) were reduced (P < .05) when compared to early blastocysts (65%), blastocysts (65%), blastocysts (66%) and advanced blastocysts (64%). Pregnancy rates from good quality Grade 1 embryos (64%) were higher (P < .05) than from Grade 2 (45%) or Grade 3 (33%) embryos. Pregnancy rates related to synchronization of estrus between donor and recipients are: -36 hrs. (before donor) (59%), -24 hrs. (61%), -12 hrs. (68%), 0 hr. (59%), +12 hrs. (61%), +24 hrs. (58%) and +36 hrs. (41%). Differences in pregnancy rate were significant for -12 hrs. (P < .01) and +36 hrs. (P < .01) recipients. Overall pregnancy rates in recipients on day 5 (48%), 5 1/2 (50%) and 6 (53%) were reduced (P < .02) when compared to day 6 1/2 (62%), 7 (63%), and 7 1/2 (66%) and 8 (62%). Pregnancy rates within each embryo stage of development were not related to synchronization with the donor cow (-36 hrs. to +36 hrs.) or the day of the recipient cycle (day 5 to 8).     

The current status and future of commercial embryo transfer in cattle

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen currently is available in only a few countries and on an extremely limited basis. As of yet, all programs involving the production of transgenic cattle are experimental in nature.     

Comparative fertility of normal and repeat-breeding cows as embryo recipients

Morphologically normal embryos were transferred surgically into uteri of normal and repeat-breeder cows at seven days post-estrus to compare embryo survival rates in the two kinds of cows. All cows were less than ten years of age and had no abnormal genital discharges, cystic ovarian follicles, or anatomical abnormalities of the reproductive tract. Normal cows had not been inseminated after last calving. Repeat-breeders had at least four infertile services within the past six months (average of 6.2 services after calving). To test fertility of repeat-breeders at synchronized estrus, 22 anatomically-normal repeat-breeders were treated by intramuscular (i.m.) injection with prostaglandin F(2)alpha (PGF(2)alpha) on day 11 of an estrous cycle (estrus = day 0) and inseminated at induced estrus; 11 cows (50%) had a normal fetus at necropsy on day 60. Twenty-three repeat-breeders and 23 normal cows were assigned as embryo recipients and treated i.m. with PGF(2)alpha to synchronize estrus. All embryo donors were normal cows. Donors were treated with FSH and PGF(2)alpha and inseminated at estrus. On day 7 after estrus, embryos were recovered nonsurgically from donors and one embryo was transferred through a flank incision to the anterior end of the uterine horn adjacent to the corpus luteum of each recipient. Recipients that did not return to estrus were necropsied at day 60. Of 28 normal and 23 repeat-breeder recipients, 23 normal cows (82%) and 16 repeat breeders (70%) were pregnant at day 60 (P=0.235). Thus, at seven days post-estrus, the maternal environment of most of these repeat-breeders was satisfactory for maintaining pregnancy.