Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5 180 ), 3.1% (9 295 ) and 1.1% (1 89 ), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.     

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes (EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at approximately 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.     

Production efficiency of Japanese black calves by transfer of bovine embryos produced in vitro

The objective of this study was to examine the production efficiency of Japanese Black beef calves after transfer of bovine embryos derived from an in vitro procedure. In vitro-produced (IVP) embryos were obtained from in vitro maturation and fertilization and in vitro development by co-culture with cumulus cells until 7 or 8 days after insemination. In vivo-developed (IVD) embryos from superovulated Japanese Black heifers and cows 7 days after artificial insemination were used as a control group. Bovine embryos were transferred nonsurgically to recipient cows on Day 7 +/- 1 of the estrous cycle. Pregnancy was diagnosed by palpation per rectum at Day 60 to 70 after estrus. Pregnancy, abortion, perinatal accident and birth rates were examined according to the origin of embryos (IVP or IVD), the number of transferred embryos (single or twin) and the storage status (fresh or frozen-thawed). In Experiment 1, production efficiency by twin transfer of fresh IVP embryos was examined. Higher pregnancy rates (52 1% vs 42 9%, P < 0.05) and birth rates (47.0% vs. 33.0%, P < 0.05) were obtained by twin transfer than by single transfer of fresh IVP embryos. Thus, the twin transfer of fresh IVP embryos was effective for production of calves, although the birth rates for single and twin transfers of fresh IVD embryos were still higher (55.5% and 76.1%, P < 0.05). But the abortion and perinatal accident rates for twin transfer of fresh IVP embryos were also significantly greater than those for single and twin transfer of fresh IVD embryos (P < 0.05). In Experiment 2, production efficiency by twin transfer of frozen-thawed IVP embryos was examined. Either single or twin transfer of frozen-thawed IVP embryos resulted in a similar pregnancy rate (41.3% vs. 46.7%, P > 0.05) and birth rate (34.1% vs. 41.1%, P>0.05). Thus, in combination with frozen-thawed IVP embryos, the twin transfer did not enhance production efficiency. In conclusion, Japanese Black beef calves could effectively produce calves by twin transfer to Holstein recipients when using fresh IVP embryos, and by single transfer when using frozen-thawed IVP embryos.     

Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.     

Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage

Bovine oocytes matured in vitro were fertilized in high proportions (92% of matured oocytes) by sperm capacitated with Ca ionophore A23187. Eight percent of inseminated oocytes that were denuded 96 h after insemination developed to the morula stage when cultured for 6-120 h after insemination with cumulus cells from the original oocytes. Inseminated oocytes denuded 96 h after insemination developed to the blastocyst stage when cultured with or without cumulus cells or in the conditioned medium from 96 h to 168-216 h after insemination (9.0%, 8.1%, and 6.8% of inseminated oocytes respectively). Six frozen-thawed blastocysts were transferred nonsurgically to 3 recipients (2 embryos/recipient). Two of the 3 recipients became pregnant, with one delivering live twins at term. Seven fresh blastocysts were transferred nonsurgically to 6 recipients (1-2 embryos/recipient). Three of the 6 recipients became pregnant, with 2 delivering live calves.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo-derived bovine embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo-derived bovine embryos (n = 10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID₅₀)/mL. Additionally, control heifers received 1.5 × 10⁶ CCID₅₀ BVDV/.5 mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.     

Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O(2), 5% CO(2), and 90% N(2). In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 microM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 microM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 microM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm(2) vs. 0.71 mm(2) surface area; n = 27, P < 0.04).     

Evaluation of virus decontamination techniques for porcine embryos produced in vitro

The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.     

Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin

Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing–thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen–thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA.     

Successful transfer of vitrified Ilama (Lama glama) embryos

The exploitation of the domestic animals species of South American camelids is of great social importance for the native people living in the High Andes. The reproductive physiology of these species is a unique challenge in the development of advanced breeding techniques. At present, the cryopreservation of embryos has not been developed and very few investigations have been conducted. The objective of the present work was to evaluate the in vivo survival of vitrified llama embryos after transfer to recipient females. Donors females were treated with a CIDR-estradiol benzoate-eCG regimen and were mated naturally 6 days after CIDR withdrawal. One ovulatory dose (8 microg) of GnRH was administered immediately after mating. A second mating was allowed 24 h later. Embryo recovery was performed nonsurgically between 8 and 8.5 days after the first mating. Twenty-two ova/embryos were recovered from 12 donor females. Hatched blastocysts were exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol (P/V)) in three steps, and after loading into 0.25 ml straws, were plunged into liquid nitrogen. For embryo transfer, recipients animals were ovulation-synchronized using GnRH administered at the same time as donors. A total of eight vitrified-warmed embryos and 12 fresh embryos were nonsurgically transferred to four and six recipient females, respectively (two embryo per recipient). The pregnancy rates were 50 and 33.3% for recipients that had received vitrified embryos and fresh embryos, respectively. The results demonstrated the effectiveness of this simple vitrification method for cryopreservation of llama embryos.     

Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.     

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

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

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.     

Normal calves produced after transfer of in vitro fertilized embryos cultured with an antiviral compound

Bovine viral diarrhea virus (BVDV) replicates in embryo co-culture systems and remains associated with developing IVF bovine embryos, despite washing and trypsin treatment. Previous research demonstrated that 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) inhibits replication of BVDV in cultured cells. The objective of this study was to evaluate the capability of IVF embryos to develop into normal, weaned calves after exposure to antiviral concentrations of DB606 during IVC. Oocytes were obtained from cows via transvaginal, ultrasound-guided follicular aspiration. Presumptive zygotes (n = 849) that resulted from fertilization of these oocytes were cultured for 7 d in medium supplemented with 0.4 microM DB606 or medium lacking antiviral agent. All blastocysts (n = 110) were transferred individually into the uterus of a synchronized recipient. The pregnancy status of recipients was determined using transrectal ultrasonography at 21-23 d after embryo transfer. Additional pregnancies as controls (n = 21) were initiated by natural breeding. Developing fetuses and resulting calves were evaluated every 27-34 d. Blastocyst development, pregnancies per transferred embryo, pregnancies maintained per pregnancies established, gestation length, gender ratio, birth weights, viability of neonates, complete blood counts, and serum chemistry profiles at 3 mo of age and adjusted 205 d weaning weights were compared for research treatments. Development to weaning after exposure to DB606 did not differ significantly from controls. In conclusion, bovine embryo cultures can be safely supplemented with antiviral concentrations of DB606; addition of DB606 agent might prevent viral transmission if BVDV were inadvertently introduced into the embryo culture system.     

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.