Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm.

Puncture of the Equine Embryonic Capsule and Its Repair In Vivo and In Vitro.

Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured in vitro for 48 hrs (n = 3 per puncture group) or transferred to recipient mares and recovered 48 hrs later (n = 3 per puncture group). No pregnancies resulted from the 10 embryos punctured with the acupuncture needle, whereas 15 of 20 (75%) and 10 of 15 (67%) punctured on the micromanipulator or manually with the microneedle resulted pregnancies. Neither acupunctured nor microneedle-punctured embryos repaired their capsules in vitro. The acupunctured embryos also failed to repair their capsule after 48 hrs in vivo and subsequent uterine flushing yielded numerous capsular vesicles. The microneedle-punctured embryos did repair their capsule in vivo. Puncture with the microneedle opens the way for development of a manual method to vitrify equine embryos.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue.

We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 microm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

Design and validation of a robotic needle positioning system for small animal imaging applications.

A needle-positioning robot has been developed for image-guided interventions in small animals. The device is designed to position a needle with an error < or =100 microm. The robot has two rotational axes (pitch and roll) to control needle orientation, and one linear axis to perform needle insertion. The three axes intersect at a single point, creating a remote center of motion. Needle positioning error was quantified at ten target locations for each rotational plane. The measured needle positioning accuracy in free space was 54 +/-12 microm and 91 +/- 21 microm for the pitch and roll axes, respectively. The device's accuracy compares favorably with the sizes of typical interventional targets in mouse models.

Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo.

Early embryonic development in the horse is characterized by the formation of an unusual acellular glycoprotein "capsule" between the trophectoderm and the overlying zona pellucida. This structure is first detected between days 6 and 7 after ovulation and completely envelops the spherical conceptus until as late as day 23 of gestation. In the present study, a micromanipulator was used to remove the capsule from 15 embryos on day 6-7 after ovulation. None of these denuded embryos developed into ultrasonographically detectable pregnancies after surgical transfer into recipient mares whereas four of six control embryos handled and transferred similarly but without capsule removal developed normally, thereby demonstrating clearly a role for the capsule in embryonic survival. In addition, observation of the embryonic investments after embryo collection and during micromanipulation led to the hypothesis that hatching of the horse embryo from its zona pellucida is assisted by the force of the expanding capsule, which causes the attenuating zona to literally burst open.

Comparison of fertility between intracytoplasmic sperm injection and in vitro fertilization with a partial zona pellucida incision by using a piezo-micromanipulator in cryopreserved inbred mouse spermatozoa.

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.

[Microdissection of chromosomes in veterinary medicine]. / Mikrosezierung von Chromosomen in der Veterinärmedizin.

Microdissection of chromosomes and subsequent amplification of the DNA material by PCR allow the development of libraries, region-specific DNA probes or chromosome painting probes. Veterinary medicine will benefit from this method in instances where clarification of numercal or structural chromosome aberrations is needed.

In vitro development of bovine embryos encapsulated in sodium alginate.

The objective of this study was to evaluate the in vitro development of bovine embryos encapsulated in alginate. Day-4 embryos produced in vitro (n = 110) were encapsulated with 1.5% sodium alginate and co-cultured with oviduct cells. Unencapsulated embryos (n = 106) were used as controls. In vitro development rate to the blastocyst stage at Day 7 was similar between encapsulated, 42.7%, (47/110) and control. 34% (36/106). embryos. Although encapsulated embryos were able to hatch on Day 9, they did so in a lower proportion than controls (P < 0.05). In conclusion, alginate encapsulation of bovine embryos does not disturb the in vitro development up to the blastocyst stage but significantly reduces the hatching process.

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture.

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.

Mechanoreceptor activity in the gills of the carp. II. Gill arch proprioceptors.

The presence of gill arch proprioceptors in the gills of a teleost, Cyprinus carpio L., is demonstrated and their firing characteristics are analysed. Spontaneous activity of gill arch proprioceptors was recorded from epibranchial ganglia. In paralysed fish the mean discharge rate for 16 receptors ranged from 11.3 impulses X sec-1 (SD 0.3) to 25.7 impulses X sec-1 (SD 0.4). The discharge rate could be influenced by displacement of the main elements of the respiratory pumping system. The receptors showed a tonic response. Their firing frequency was approximately linearly related to gill arch position and, hence, showed a respiratory modulation during ventilation. Gill arch adduction caused a decrease and abduction an increase in discharge rate. In actively breathing fish the mean firing frequency of 30 neurons ranged from 9.9 impulses X sec-1 (SD 0.7) to 40.1 impulses X sec-1 (SD 6.7). These gill arch proprioceptors are located in the cartilaginous strip between the epibranchial and the ceratobranchial of each gill arch and are innervated by the pretrematic branches of the vagal branchial nerves. The role these proprioceptors play in the regulation of gill movements during both feeding and ventilation is discussed.

[Production of chimeras by embryo microsurgery and their possible application in cattle breeding]. / Erstellung von Chimären durch Embryo-Mikrochirurgie und deren mögliche Bedeutung für die Rinderzucht.

An important contribution to genetic and embryonic engineering in mammalian embryos is the production of chimeric mammals. Chimeras can be produced by aggregating the blastomeres of two different embryos to make a single individual. A method is described to produce bovine chimeras by aggregating non-surgically collected embryos of different breeds and transferring the aggregated embryos nonsurgically to synchronised recipients. An overt chimeric bull calf (Holstein-Friesian and Brown-Swiss) was born on March 31st, 1984. Chimeras have many uses in research, but one practical use is to rescue defective embryos that would not develop to term on their own. Such embryos can be aggregated with normal blastomeres to produce a chimera. Some of the initially defective cells can even become part of the germ line in the chimera and give rise to perfectly normal eggs or sperm. Two models for using this technique in cattle research are described. The lethal disorder arachnomelie has a rather high frequency in some German Brown-Swiss populations. Chimeric calves developing from embryos descending of carrier matings could help to find out the genetic and developmental background of this disorder. Chimeras produced of transgenic teratocarcinoma cell lines and normal embryos offer one possibility to produce transgenic cattle. The advantages and disadvantages of these possibilities are discussed.