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.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Interspecies embryo and nuclei transfer as an approach to endangered mammalian species conservation

A review represents the current status in the area of interspecies embryos and nuclei transfer for conservation of mammalian species and breeds. Modern a chievements have been reviewed in a historical pretext and key techniques, main problems, and perspectives of this approach have been discussed.     

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.     

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.     

The use of lead as an ionic tracer for investigating routes of passive fluid transfer

Several inorganic ions, including lead, barium, silver, and thallium, have been tested as possible tracers for demonstrating fluid-accessible channels in functional epithelia at the ultrastructural level. The most useful of the ionic tracers examined was the lead (plumbous) ion, administered for short time intervals (less than 2 min) and "captured" with phosphate used as the buffer in the fixative. Passive fluid and ion-accessible channels of rat parotid salivary gland have been examined with this method. At short tracer infusion times (0.5-1.0 min), localization of the tracer was primarily extracellular, although intracellular deposits were observed in the following sites: smooth membrane-delimited endocytic vesicles of both epithelial and connective tissue cells, inner Golgi cisternae, and occasional cisternae of rough endoplastic reticulum. The lead tracer readily penetrated tight junctions between parotid acinar cells but rarely passed through the tight junctions between intercalated duct cells and did not penetrate junctions of striated duct cells. Fat cells observed in the stroma of this gland were the only cells that exhibited lead tracer in the cytosol, suggesting that the plasmalemma of this cell type is more permeable to exogenous ions than the plasmalemma of other cell types present in this gland.     

The current status of equine embryo transfer

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.     

Pregnancy established in an infertile patient after transfer of a donated embryo fertilised in vitro.

In vitro fertilisation after stimulation of the ovulatory cycle has led to successful pregnancy. If more oocytes are recovered than are needed they may be left unfertilised, preserved, or donated to a recipient couple from whom oocytes cannot be obtained. A case of human pregnancy initiated by transfer of a donated embryo fertilised in vitro is reported. The donor was a 42 year old woman with primary infertility from whom six follicles were aspirated after stimulation of the ovulatory cycle. The recipient was a 38 year old infertile woman who had undergone several unsuccessful attempts for artificial insemination from a donor. Five oocytes were recovered from the donor''s six follicles, four of which were inseminated with spermatozoa of the donor''s husband and the fifth with a frozen sample of semen. Three of the four embryos fertilised by her husband were returned to the donor and the fifth was transferred to the recipient. No pregnancy was recorded in the donor, but pregnancy was confirmed in the recipient, though spontaneous abortion occurred after 10 weeks. This case will give useful information for further study of in vitro fertilisation, but also raises many ethical issues.     

Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows

Objectives were to determine progesterone concentration (P4) from days 4 to 28 relative to presumptive estrus necessary for maintenance of pregnancy in lactating Holstein cows. Cows were assigned to the low P4 (LowP4, n = 28) or control (n = 153) treatments. All cows were presynchronized with two injections of PGF_2α (14 d apart) and an ovulation synchronization protocol (11 d later; GnRH on day −10, PGF_2α on day −3; and GnRH on day 0 = presumptive estrus). Cows in the Low P4 treatment received 2 injections of prostaglandin F_2α on days 4 and 5 (day 0 = presumptive estrus) and a new CIDR insert on day 5 that was replaced every 7 d until day 28. Blood was sampled on days −9, −2, 0, 4, 7,14, 21, and 28. Ovaries were examined with ultrasound on days −9, −3, and 7 and cows bearing a corpus luteum ≥20 mm on day 7 received an embryo. On days 0, 4 and 7 P4 did not differ (P ≥ 0.27) but control cows had greater (P < 0.01) P4 on days 14, 21, and 28. Progesterone concentration fold change from day 0 to 7 was not (P = 0.14) affected by treatment, but P4 concentration fold change from day 7 to 14 was (P < 0.01) greater for control cows compared with LowP4 cows. No LowP4 cows became pregnant after embryo transfer, whereas 35.7, 25.5, and 21.4% of control cows were pregnant on day 28, 42, and 63, respectively. Progesterone concentration fold changes from day 0 to 7 (P = 0.03) and from day 7 to 14 (P = 0.05) were associated with pregnancy outcomes on day 63. Among cows that were pregnant on day 63, the minimum P4 concentration fold changes from day 0 to 7 and from day 7 to 14 were 2.71 and 1.48, respectively. Interestingly, cows with P4 concentration <5 ng/mL on day 14 were (P = 0.01) and tended to be (P = 0.07) more likely to lose pregnancy from day 28 to 42 and from day 28 and 63, respectively. Faster rise in P4 concentration during the metestrus and early diestrus are associated with pregnancy establishment following embryo transfer, which suggests that early rise in P4 concentration has an indirect effect on embryo development through modulation of uterine environment and secretion of histotroph. Furthermore, the positive effects of early rise in P4 concentration appear to go beyond the phase of maternal recognition of pregnancy through adhesion and placentation stages.     

The Holstein cow in embryo transfer today as compared to 20 years ago

Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased (P < 0.05) in one program over time, but not in the other three. Changes in the type of cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates (P < 0.001) following transfer of embryos into Holstein heifers than into lactating dairy cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease (P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.     

Cattle breed as a source of variation in embryo transfer

The records of 1596 embryo collections were retrospectively analysed to investigate the influence of breed on embryo production and pregnancy rates. The breed of the donor cow was a significant source of variation in the results of embryo transfer. Total embryo production per collection ranged between breeds from 6.0 to 16.2, number of transferable embryos from 2.8 to 6.6 and percent transferable from 37% to 68%. The percent of pregnancies per collection ranged from 2.0 to 4.0 and pregnancy rates from 37% to 75%. The percent of transferable embryos and the pregnancy rate was independent of the total embryos collected. Care should be taken when interpreting the breed differences, since the donor cows were not randomly representative of the breeds and were selected on different bases. Brangus (16.2), Simbrah (15.8) and Beefmaster (13.0) produced the most embryos. The most transferable embryos were collected from Simmentals (6.6), Brangus (6.6), Chianina (6.2), Beefmaster (6.1), Simbrah (6.1) and Saler (6.0) cows. Angus (68%) had the highest percent transferable, followed by Saler (64%), Chianina (60%), Limousin (52%) and Simmental (51%). Pregnancy rates were highest in the Herefords (75%), Saler (57%), Zebu (56%), Charolais (53%), Longhorns (52%) and Simbrah (50%). Estrus response to superovulation varied between breeds, but this did not account for all the breed differences in embryo production.     

The use of embryo transfer and steroid hormone replacement therapy in the study of prenatal mortality

Embryo transfer and steroid hormone replacement therapy have been used to advantage in studies on prenatal mortality, particularly in sheep.The form, timing and magnitude of loss, have been well documented and steroid hormone replacement therapy coupled with embryo transfer has clearly shown the involvement of the ovarian steroid hormones secreted before, during and after estrus in survival and development of embryos.However, little is known of the manner in which the steroid hormones exert their effects on the environment of pre-implantation embryos; a stage when most mortality occurs.     

The embryo as allograft

The main problems and experimental results with respect to the mechanism by which the embryo, as "allograft", is tolerated within the maternal organism are reviewed. The following hypotheses are discussed: the immunological "immaturity" of the embryo and of its appendages; the immunological incompetence of the maternal organism; the uterus as an immunologically "privileged" area; the existence of an immunological barrier between the mother and the embryo.     

Lack of risk of transmission of caprine arthritis-encephalitis virus (CAEV) after an appropriate embryo transfer procedure

The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.