Non-surgical recovery of bovine embryos

A simple one-way cannula technique for non-surgical recovery of bovine embryos has been described. The collector was very reliable, stable, and easy to use. It could be guided through cervix and without damage to the genital tract into both uterine horns on all attempts. Ninety-six per cent (96%) of the flushing medium was recovered. Bleeding occurred rarely. The post-flushing reproductive performance was satisfactory. An average of 4.1 eggs per dairy cow were recovered yielding a recovery rate of 46%. The method was performed under farm conditions with and without the use of a chute or stanchion. Several general practitioners have used the method with success after a brief training period. Good manipulative skills were a prerequisite for good results.     

The viability of deep-frozen cow embryos

Day 7 cow embryos were frozen in 1.5 M-DMSO in PBS at 0.3 degrees C/min to -36 degrees C and at 0.1 degrees C/min between -36 and -60 degrees C before being plunged directly into liquid nitrogen. They were subsequently thawed (rapidly to -50 degrees C, at 4 degrees C/min from -50 to -10 degrees C, and rapidly again) to room temperature. Embryonic viability was tested by four different transfer techniques. Maximum pregnancy rate (8/12) was obtained with surgical transfer immediately after thawing and dilution of DMSO.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

A paracervical method for non-surgical transfer of bovine embryos

Seventy-six day-8 bovine blastocysts were transferred by a paracervical non-surgical process to determine its feasibility as an alternative method of bovine embryo transfer. Forty-seven of the 76 transfers resulted in pregnancy (62%), as determined by rectal palpation at 45 to 90 days of gestation. This indicates that embryo transfer via the paracervical method is a viable alternative to surgical and non-surgical transfer in cattle.     

Developmental kinetics of bovine nuclear transfer and parthenogenetic embryos

Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.     

Non-surgical recovery of bovine eggs.

An improved, non-surgical technique for recovering bovine eggs is described using modified Foley catheters. Egg recovery rates per attempt were, $\text{36}\text{51}$ (71%), for normal, unsuperovulated donors and $\text{4}\text{38}$ (11%), for unsuperovulated donors with known fertility problems. For superovulated donors, eggs were collected in $\text{24}\text{26}$ attempts (92%) averaging 6.9 per recovery.     

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.     

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.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos

One impediment to the more widespread use of freezing as an adjunct to embryo transfer in cattle has been the method used to remove the protective compound from the thawed embryos. Recently, a method has been developed that permits bovine embryos to be diluted out of the protective solution within the plastic straw in which the embryos were originally rrozen and thawed. The method requires only about 10 minutes to perform and does not require a microscope or other laboratory equipment. Therefore, frozen bovine embryos can be thawed, diluted, and transferred nonsurgically into recipient cattle under conditions quite similar to those used for artificial insemination. A large number of field trials of this method have been performed during the past 2 1 2 years. A total of 327 pregnancies have been established by the nonsurgical transfer of 1259 embryos that had been frozen, thawed, and diluted by this method.     

Prevention of bovine herpesvirus-1 transmission by the transfer of embryos disinfected with recombinant bovine trypsin

This study deals with the potential for the introduction of infectious agents through the use of animal-derived products. The efficacy of a recombinant bovine trypsin (RBTr) as a replacement for porcine pancreatic trypsin and a disinfectant for bovine herpesvirus-1 (BHV-1)–infected embryos was investigated according to the sanitary guidelines of the International Embryo Transfer Society. Treatment of in vivo and in vitro fertilized embryos contaminated with BHV-1 (10⁵ TCID50/mL) in the presence of RBTr (525 U/mL) for 120 s, effectively removed the infectious virus compared with untreated and washed embryos (P < 0.05). Transfer of in vivo fertilized and disinfected embryos to BHV-1 seronegative recipients (n = 24) resulted in 14 pregnancies and 11 calves born free of BHV-1. In contrast, transfer of unwashed or undisinfected embryos to four recipients resulted in seroconversion and no pregnancies at term. It was concluded that the use of RBTr could be considered as an alternative method of rendering embryos free of BHV-1 and thus reduce the potential risk of disease transmission to embryo recipients and offspring.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.