Use of a rapid method of cryopreservation of bovine embryos for non-surgical transfer in transplantation practice

The embryos were frozen and thawed in Cassou minipaillette by a rapid method. Embryos with cryoprotective agent (glycerol, 1.5 M) were placed directly into the freezing medium at the temperature of -6 to -7 degrees C, frozen after seedling at the temperature decrease by 0.3 to 0.5 degrees C per minute to the temperature of -32 degrees C and then transferred directly into liquid nitrogen. They were thawed in a bath warm 20 to 37 degrees C. After thawed the cryoprotective agent was evacuated in 1.1 M sucrose. The best-quality embryos were selected for freezing. Out of these 366 thawed so far, with average survival of 74.31%. The total of the 268 thawed embryos were transferred ipsilaterally, by a non-surgical method, to 190 synchronised heifers, out of which 105 (55.26%) got in calf. Rapid freezing method based on 1.5 M of glycerol and thawing at the presence of 1.1 M sucrose proved effective and suitable for practice, as not only sufficient reviviscence of embryos and their survival in womb are guaranteed, but also a substantial shortening of the freezing as well as thawing process.     

Non-surgical transfer of bovine embryos

Bovine embryos obtained from donors six to nine days after oestrus were transferred non-surgically at a rate of one per recipient using a sterile insemination instrument, protected from contamination by the vagina with a plastic sheath. The percentage of recipients pregnant increased with the age of embryo transferred and for day 6 and 7 embryos was 33% compared to 58% for day 9 and 8 embryos. This difference approached statistical significance. Bacterial contamination of the instrument on withdrawal after transfer was not related to the success or failure of pregnancy. Maintenance of pregnancy to term and calving appeared to be normal. It is suggested that this method could be used for the routine transfer of eight and nine day embryos.     

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H₂O₂ and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H₂O₂ (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.     

Increase in calf production by the transfer of bisected bovine embryos

A total of 132 embryos were recovered from 17 superovulated donor cows 7 d after estrus. Seventy-four embryos were selected and assigned to 2 treatment groups. The number of whole embryos that were directly transferred (Group A) and bisected (Group B) were 44 and 30 embryos, respectively. Sixty demi-embryos were produced from 30 morulae to blastocyst-stage embryos that were bisected. One hundred-three embryos, including whole and demi-embryos without zonae pellucidae, were nonsurgically transferred. Only one whole or demi-embryo was transferred to each recipients. The pregnancy rate for whole embryos (A) was 63.6% (28/44), while for demi-embryos (B) it was 74.6% (44/59). There was no significant difference between the pregnancy rates of whole embryos (A) and bisected embryos (B) transferred 7 d after estrus. Forty-three calves including the 14 sets of identical twins were obtained from 30 original embryos (143.3%) using the embryo bisection technique.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.     

体细胞核移植生产转基因牛胚胎的研究

为了优化利用体细胞核移植生产转基因牛早期胚胎的体系,以携带绿色荧光蛋白-新霉抗性双标记基因的pMSCV质粒转染的胎牛耳成纤维细胞为供体,以体外成熟的牛卵母细胞为受体构建克隆胚,研究供体细胞的传代次数对转基因胚的影响和重构胚在不同电场条件下（电场强度、直流电脉冲次数）融合效率及其在不同体外培养系统中的发育效果。结果表明：传代5次的细胞做核供体较有利于转基因胚的发育;在电场条件为电场强度1.6 kV/cm、直流脉冲1次的融合率最高;负压单层细胞共培养体系中的转基因囊胚发育率较好,与未转基因体细胞核移植胚的发育相比无显著差异（P〉0.05）。     

Cryopreservation of bovine embryos

The method of cryopreservation of embryos aged seven days, proposed for embryo transfer with cattle by Niemann (1985), was tested under production conditions on three cattle breeding farms and three experimental animal units. The number of donors was 128, and 467 intact embryos were obtained from them and were cryopreserved in semen straw. Following thawing, 455 were recovered, and 439 (96.5 percent) of these were suitable for transfer. A pregnancy rate of 49.0 percent was recorded from 412 transfers. This rate was differentiated by oestric cycle conditions of heifer recipients, which gave percentages of 46.0 among recipients of seven-day old embryos, 45.7 for eight-day recipients, and 65.8 for six-day recipients. Related to pregnancy results recorded on the same units from transfer of freshly collected seven-day embryos, the efficiency coefficient was 0.69 (550 fresh transfers = 65.4 percent and 222 cryopreserved transfers = 48.2 percent). The method is recommended for general field practice.     

Assisted hatching of poor-quality bovine embryos increases pregnancy success rate after embryo transfer

Embryos of good, fair and poor quality were collected from superovulated cows and subjected to zona cutting (ZC) treatment using a needle under either an inverted microscope or a stereomicroscope. One (single transfer) or 2 (twin transfer) embryos with or without prior ZC treatment were transferred nonsurgically to recipients. Without the ZC treatment, lower embryonic quality resulted in lower pregnancy success rates. However, the ZC treatment increased the pregnancy success rate following transfer of poor-quality embryos, but not the pregnancy rate after transfer of good- or fair-quality embryos. No differences were observed between the pregnancy success rates after the transfer of embryos treated under the inverted microscope and those after transfer of embryos treated under the stereomicroscope, and this was the same after single and twin transfer. Moreover, ZC treatment of embryos prior to transfer did not result in an increased abortion rate, irrespective of the number of transferred embryos. In conclusion, ZC treatment improves pregnancy success rates following transfer of poor-quality embryos. Moreover, the results indicate that ZC treatment by using a stereomicroscope is practical for on-farm application.     

Some aspects of non-surgical embryo transfer in cattle

Non-surgical embryo recovery methods in cattle produce recovery rates similar to those obtained by surgical techniques. The average pregnancy rate following non-surgical transfers, (30%), is however much lower than that obtained following surgical transfer methods (60%).From the literature there is clear evidence of the existence of a luteolytic factor produced by the uterus and also some indication of a luteotropin which counteracts the luteolytic activity during pregnancy. Luteolysis of the corpus luteum is produced via the utero-ovarian pathway by the luteolytic factor present in the blood draining from most of the endometrium of the ipsilateral horn. The nature and mechanism of action of the luteotropic factor are not yet known but in order to counter the luteolytic effect, it must also exert its action in the ipsilateral horn.The unilateral relationship of the conceptus and the ovary bearing the corpus luteum explains the importance of embryo siting in determining the survival rate of embryos. After embryo deposition into the tip of the ipsilateral horn the antiluteolytic action of luteotropin is available either for local action or for conveyance to the corpus luteum. After embryo transfer to the contralateral horn or base of the ipsilateral horn the luteotropic factor has to travel via the uterine body down the ipsilateral horn in order to exert its influence. It is therefore concluded that embryos sited in the tip of the ipsilateral horn are more likely to survive than those at other locations which will require the transport of luteotropin produced in optimal quantities.Suggestions for further research to improve pregnancy rates following non-surgical methods are discussed.

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Kurzfassung Die Anwendung nicht-chirurgischer Embryoerhaltungsverfahren bei Tieren führt zu Gewinnungsquoten, die denen von chirurgischen Verfahren entsprechen. Die durchschnittliche Trächtigkeitsquote von 30% nach nicht-chirurgischer Verlagerung ist viel niedriger als die bei chirurgischen Verlagerungsverfahren erreichten 60%.Die Fachliteratur hat die Existenz eines im Uterus erzeugten luteolytischen Faktors nachgewiesen und enthält einige Anhaltspunkte für ein Luteotropin, das der luteolytischen Tätigkeit während der Trächtigkeit entgegenwirkt. Die Luteolyse des Corpus luteum wickelt sich über den Uterus-Ovarial-Weg ab, über den das Blut mit dem luteolytischen Faktor aus dem Grossteil des Endometriums der ipsilateralen Tuba abläuft. Die Art des luteotropischen Faktors und der Mechanismus seiner Tätigkeit sind noch nicht bekannt; um jedoch die luteolytische Wirkung erfolgreich zu unterbinden, muss er seine Wirkung auch in der ipsilateralen Tuba ausüben, von der der luteolytische Faktor in das das Corpus luteum enthaltende Ovarium abgegeben wird.Die Einseitige Beziehung zwischen Fetus und Corpus luteum enthaltendem Ovarium erklärt die Bedeutung der Embryolagerung bei der Bestimmung der Überlebensrate von Embryos. Die antiluteolytische Wirkung von Luteotropin ist einzusetzen entweder als Wirkung an Ort und Stelle oder als Weiterleitung in das Corpus luteum, nach Embryoeinlagerung in die Spitze der ipsilateralen Tuba. Nach Embryoverlagerung in die kontralaterale Tuba, oder die Tubenbasis, muss der luteotropische Faktor über den Uteruskörper in die ipsilaterale Tuba hinunter verlagert werden, um eine entsprechende Wirkung auszuüben. Daher wird gefolgert, dass in der Spitze der ipsilateralen Tuba gelagerte Embryos die besten Überlebenschancen im Vergleich zu denjenigen in anderen Lagen haben, bei denen eine Weiterleitung des in optimalen Mengen erzeugten Luteotropins erforderlich ist.Anregungen für weitere Forschungstätigkeit zur Verbesserung der Trächtigkeitsquoten nach nicht-chirurgischen Verfahren werden erörtert.

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Resume L'application des méthodes non-chirurgicales de récupération de l'embryon chez les bovins donne des taux de récupération analogues à ceux des techniques chirurgicales. Le taux moyen de gestation de 30% à la suite de transferts non-chirurgicaux est nettement inférieur aux 60% obtenus suivant les méthodes chirurgicales de transfert.La littérature établit clairement l'existence d'un facteur lutéolytique utérin et donne certaines indications de l'existence d'une lutéotropine qui contrarie l'activité lutéolytique pendant la gestation. La régression du corps jaune s'effecture via le canal utéro-ovarien qui draine le sang, contenant le facteur lutéolytique, de la plus grande partie de l'endomètre de la corne utérine homolatérale. La nature du facteur lutéotropique et le méchanisme de son action ne sont pas encore connus, mais pour que ce facteur neutralise l'effet lutéolytique, il faut qu'il exerce aussi son action dans la corne utérine homolatérale d'où le facteur lutéolytique est drainé vers l'ovaire contenant le corps jaune.La relation unilatérale du produit de la conception et de l'ovaire contenant le corps jaune explique l'importance de l'emplacement de l'embryon pour le taux de survie des embryons. L'action anti-lutéolytique de la lutéotropine s'exerce a l'emplacement de l'embryon soit localement soit par déplacement vers le corps jaune à la suite du dépôt de l'embryon au sommet de la corne utérine homolatérale.Après le transfert de l'embryon dans la corne contra-latérale ou base de la corne, le facteur lutéotropique doit être transporté via le corps utérin vers la corne utérine homolatérale pour exercer la même influence. La conclusion est donc que les embryons placés dans la pointe de la corne utérine homolatérale ont plus de chances de survie que ceux placés en d'autres endroits, et qui nécessiteront le transport de lutéotropine produite en quantités optimales.Des propositions de recherches complémentaires en vue d'améliorer les taux de gestation suivant des méthodes non-chirurgicales sont examineés.

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Riassunto L'applicazione di metodi non chirurgici per il trapianto embrionale nei bovini si traduce in tassi di recupero simili a quelli ottenuti con tecniche chirugiche. Il tasso di gravidanza medio del 30% a seguito di trapianti incruenti è molto più basso del 60% che si ottiene con metodi di traslazione chirurgici.La letteratura dimostra chiaramente l'esistenza di un fattore luteolitico di produzione uterina; vi sono pure indicazioni della presenza di una luteotropina, che contrasta l'attività luteolitica durante la gravidanza. La luteolisi del corpo luteo ha luogo attraverso il dotto utero-ovarico, che permette il drenaggio del sangue contenente il fattore luteolitico da gran parte dell'endometrio del corno omolaterale. Natura e meccanismo d'azione del fattore luteotropico sono tuttora ignoti, ma se l'effetto luteolitico deve essere contrastato con successo, l'azione di tale fattore deve esplicarsi anche nel corno omolaterale, dal quale il fattore luteolitico viene drenato all'ovaia, che reca il corpo luteo.Il rapporto unilaterale fra concepito ed ovaia recante il corpo luteo spiega l'importanza della posizione dell'embrione per il tasso di sopravvivenza di quest'ultima. L'azione antiluteolitica della luteotropina si esplica sia localmente, sia per convogliamento al corno luteo dopo la deposizione dell'embrione nella punta del corno omolaterale. Dopo la spostamento dell'embrione al corno controlaterale, o alla base dello stesso, il fattore luteotropico deve essere convogliato tramite il corpo dell'utero al corno omolaterale onde esplicare analogamente la propria azione. Si conclude pertanto che gli embrioni situati nella punta del corno omolaterale hanno maggiori probabilità di sopravvivenza di quelli situati altrove, che abbisognano del convogliamento, in quantità ottimali, della luteotropina prodotta.Si discutono proposte di ulteriori ricerche tendenti a migliorare i tassi di gravidanza con l'applicazione di metodi non chirurgici.

     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

In vitro developmental potential of bovine nuclear transfer embryos derived from primary cultured cumulus cells

The in vitro development and the quality of blastocysts produced from the nuclear transfer (NT) embryos reconstituted from primary cultured cumulus cells (NT-cumulus) were examined compared to in vitro fertilized embryos (IVF) and NT embryos reconstituted from the embryonic blastomeres (NT-blastomere). The cleavage rate, and the development to blastocyst were the same for all three sets of embryos. The time required for blastocoel formation starting from the time of the initial cleavage was shorter for NT embryo groups than IVF ones. All experimental groups produced morphologically similar and normal blastocysts containing the same cell number. The percentage of the blastocysts with normal chromosomal complements were the same for NT-cumulus and IVF.     

Infection of early bovine embryos with bovine herpesvirus-1

Recently hatched bovine embryos were exposed in vitro to 1 of 4 strains of bovine herpesvirus-1 to determine whether the viruses would replicate in these embryos and, if so, what pathologic consequences would ensue. Exposure to each of the viruses resulted in embryonic infection and death, and replication of the agents was demonstrated by electron microscopy and titration of progeny virus. There were no dramatic differences between virus strains in pathogenicity or in the ultrastructural pathologic findings of infection.     

Microtubule distribution in somatic cell nuclear transfer bovine embryos following control of nuclear remodeling type

This study was conducted to evaluate the microtubule distribution following control of nuclear remodeling by treatment of bovine somatic cell nuclear transfer (SCNT) embryos with caffeine or roscovitine. Bovine somatic cells were fused to enucleated oocytes treated with either 5 mM caffeine or 150 µM roscovitine to control the type of nuclear remodeling. The proportion of embryos that underwent premature chromosome condensation (PCC) was increased by caffeine treatment but was reduced by roscovitine treatment (p < 0.05). The microtubule organization was examined by immunostaining β- and γ-tubulins at 15 min, 3 h, and 20 h of fusion using laser scanning confocal microscopy. The γ-tubulin foci inherited from the donor centrosome were observed in most of the SCNT embryos at 15 min of fusion (91.3%) and most of them did not disappear until 3 h after fusion, regardless of treatment (82.9-87.2%). A significantly high proportion of embryos showing an abnormal chromosome or microtubule distribution was observed in the roscovitine-treated group (40.0%, p < 0.05) compared to the caffeine-treated group (22.1%). In conclusion, PCC is a favorable condition for the normal organization of microtubules, and inhibition of PCC can cause abnormal mitotic division of bovine SCNT embryos by causing microtubule dysfunction.     

Nuclear transplantation in bovine embryos

This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.     

Production and quality of bovine oocytes and embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.