Reliability of ultrasonographic measurements of bovine sole structures in relation to sole horn thickness, measured by computed tomography, and sole horn hardness.

The goal of the present study was to determine the effect of sole horn thickness (SHT) and sole horn hardness (SHD) on ultrasonographic visualization of sole structures in the inner and outer claws of 150 Holstein-Friesian cows, and to evaluate different ultrasound frequencies for this purpose. Ultrasonographic views of the sole structure were considered complete when 3 echogenic lines, representing the ventral surface of the sole horn, the borders of the sole horn and soft-tissue layer, and the ventral surface of the distal phalanx, were seen. The proportion of complete ultrasonographic views of the sole structures, designated as the ultrasonographic visualization proportion (UVP), and the measurement errors of SHT were evaluated by comparing images from computed tomography (CT) and ultrasonography. The latter images were generated using 3 different probes, frequencies of 6.5 and 5.0 MHz, and 2 different ultrasound machines (#1 and #2) to assess the apex, middle, and heel regions of the claws. The UVP were 60.8 to 77.9% for the 6.5-MHz probe in ultrasound machine #1 (probe A), which were lower than those (>90%) for both the 5.0-MHz probe in ultrasound machine #1 (probe B) and the 5.0-MHz probe in ultrasound machine #2 (probe C). The UVP was significantly lower in claws with an SHD ≥50 units than in claws with an SHD <40 or 40 to <50 units (UVP: 77.1% compared with 93.7 and 91.4%, respectively) when measured with probe B. In claws with an SHT <10 mm, the UVP was significantly lower when SHD was ≥50 units compared with <40 or 40 to >50 units; the values were 69.0% versus 91.3 and 85.9%, respectively, for probe A, and 89.7% versus 100 and 100%, respectively, for probe B. When SHT were measured by either probes A or B in ultrasound machine #1, the proportions of claws in which ultrasonographic values were within a ±1 mm range compared with the values obtained by CT were 84.9 to 91.3% for CT-determined SHT <5 mm, 66.7 to 71.9% for CT-determined SHT 5 to <7 mm, 28.9 to 51.2% for CT-determined SHT 7 to <10 mm, and 6.2 to 19.7% for CT-determined SHT ≥10 mm. The data indicated that increased SHT was associated with a decrease in ultrasonographic measurement accuracy. In claws with an SHT <5 mm, the high proportion of ultrasonographic values that were accurate within a ±1 mm range suggests that this imaging modality would be useful in cows with thin soles.

Evaluation of follicular development and oocyte quality in pre-pubertal cats.

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre-pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre-pubertal cats with estimated age of <20, 20-40 and 100-120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre-pubertal cats with 100-120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre-pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre-pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre-pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes.

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.

Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Modulation of reflex responses during fictive locomotion in the forelimb of the cat.

During cat forelimb fictive locomotion, short-latency reflex pathways were examined by recording nerve discharges and intracellularly from motoneurones. Stimulation of cutaneous afferents, superficial radial nerves, evoked trisynaptic excitation of the elbow flexors, biceps brachii and brachialis, and stimulation of muscle afferents, deep radial nerves, evoked oligosynaptic, i.e. monosynaptic and disynaptic excitation of the flexors. The short-latency excitatory postsynaptic potentials (EPSPs) evoked from both nerves were rhythmically modulated; they were facilitated during the flexion phase and suppressed during the extension phase. Stimulation of high threshold muscle afferents evoked EPSPs with a central delay of ca. 4.2 ms, which were depressed throughout episodes of fictive locomotion. Since the short-latency EPSPs and longer-latency EPSPs in the same motoneurone were differently influenced during fictive locomotion, the effects observed could not be explained by changes occurring at only the motoneuronal level but they probably occurred at the premotoneuronal level. In addition, short-latency cutaneous excitation of the distal muscles, innervated by the median and ulnar nerves, was little modulated during fictive locomotion.

Cervical interneurones oligosynaptically excited from primary afferents and rhythmically active during forelimb fictive locomotion in the cat.

In C6-C7, activity of neurones oligosynaptically excited from primary afferents was examined during fictive locomotion. Two kinds of neurones were found: neurones with activity modulated and with activity not modulated. The minimum linkage of the pathway to modulated neurones was mono- and disynaptic from muscle and cutaneous afferents, respectively, while non-modulated neurones were monosynaptically excited from both afferents. Modulated neurones were mainly located in the ventral part of the dorsal horn and adjoining intermediate region, non-modulated ones located in the dorsal horn. Modulated neurones were found near motor nuclei as well. Active periods of modulated neurones were widely distributed in the step cycle.

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro.

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation.

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.

Post-implantation development of demi-embryos and induction of decidual cell reaction in mice.

Mouse demi-embryos that developed from bisected morulae were transferred to recipients. The eu-blastocysts (distinct inner cell mass and well-developed trophectoderm) contained cells equal to 51% of the controls that developed from zona-free morulae. The rate of decidual cell reaction induced by the eu-blastocysts was not significantly different from that of the controls, but the size of the deciduum containing the egg cylinder was significantly smaller on Day 5.5 of pregnancy (P < 0.001). A significant increase in embryonic loss was observed from Day 7.5 to Day 9.5 in the eu-blastocysts (P < 0.05), while the controls exhibited no significant difference. Although the embryos from the eu-blastocysts showed retardation of developmental stages and decreased size, they attained normal stages and size regulation up to 90% of that of the control on Day 10.5. The pseudo-blastocysts (poorly developed inner cell mass enclosed by trophectoderm) contained cells equal to 25% of those of the controls and showed less than a 10% developmental rate to the egg cylinder stage. The trophectodermal vesicles (no enclosed cells) and nonintegrated forms (disorganized clusters of cells) contained cells less than 18% of those of the controls. They showed lower rates of decidual cell reaction than those in the controls (P < 0.05), and no egg cylinder was found in the deciduum. The results indicate that a severe decrease in the number of embryonic cells affects the regulation of embryonic development and decidual cell reaction in the uterus.

Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C.

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.

Stimulation of the development of bovine embryos by insulin and insulin-like growth factor-I (IGF-I) is mediated through the IGF-I receptor.

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol. Dose-response studies showed that insulin (0.5 to 10 microg/ml) and IGF-I (2 to 200 ng/ml) stimulated the development of bovine embryos to the morula stage 5 d after in vitro fertilization. The addition of 0.5 microg/ml insulin or 2 ng/ml IGF-I to the mSOF had beneficial effects on embryonic development to the morula stage in the presence of amino acids, but insulin and IGF-I did not affect the development of bovine embryos to the morula stage in the absence of amino acids. The antiIGF-I receptor antibody (alphaIR-3) completely blocked the stimulation of development to the morula stage by insulin and IGF-I. These findings suggest that the stimulation of embryonic development by insulin and IGF-I is mediated through the IGF-I receptor.

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos.

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose.

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.

Recovery, morphological quality, and in vitro maturation of follicular oocytes from bitches with pyometra.

The objective of this study was to collect oocytes from ovaries of bitches with pyometra and to characterize the quality of the oocytes recovered. In 10 of 12 cases of pyometra, follicles with a diameter of 500 microm to 1mm were observed in the ovaries. A total of 710 oocytes were collected from 10 bitches by puncturing individual follicles after slicing the ovarian tissues. Oocyte recovery was successful from a bitch with severe clinical signs of pyometra. Of the oocytes collected, 53.5% were surrounded by > or =2 layers of cumulus cells, and 55.0% of these cumulus-oocyte complexes (COCs) had a darkly pigmented ooplasm >110 microm in diameter (large-dark COCs). The number of large-dark COCs per bitch varied from 1 to 72. A germinal vesicle with fine filaments of chromatin (Type A) was observed in 51.8% (range 21.1-100%) of the oocytes of large-dark COCs. Out of 50 oocytes cultured for 72 h, 6.0% developed to Metaphase II. In conclusion, there were many follicles with a diameter of 500 microm to 1mm in ovaries of bitches with pyometra, and many oocytes recovered from these follicles underwent meiotic maturation in vitro. The number of oocytes and COCs, and the morphological quality of the germinal vesicles varied among individual bitches.

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification.

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture.

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.

Attachment, outgrowth, invasion and formation of the egg cylinder in mouse half embryos in vitro.

To investigate the effect of the decrease in the number of embryonic cells on the development and morphogenesis of mouse embryos at the peri-implantation stage, half embryos which developed from bisected morulae were morphologically classified into eu-blastocysts (eu-blasts), pseudo-blastocysts (pseudo-blasts), trophectodermal vesicles (TVs) and non-integrated forms (NIFs) and then cultured on plastic substratum, uterine-epithelial-cell monolayer and type I collagen gel. When half embryos were cultured on plastic substratum and cell monolayer, the rates of attachment and trophoblast outgrowth in the eu- and pseudo-blasts were not significantly different from those of the controls. The TVs and NIFs showed significantly lower rates of outgrowth than the controls (P < 0.01). When half embryos were cultured on type I collagen gel, no significant difference was observed in the rate of primitive endoderm formation between the eu-blasts and controls after 36 hr of culture. In the eu-blasts, however, the developmental rate to the egg cylinder at stage 8 was significantly lower than in the controls after 72 hr (P < 0.05). The pseudo-blasts revealed significantly lower rates of endoderm formation and development to the egg cylinder than the controls after 36 and 72 hr, respectively (P < 0.05). In the TVs and NIFs, the rates of outgrowth were significantly lower than those of the controls (P < 0.05) and no egg cylinder was observed. The invasion of type I collagen gel by the cytoplasmic protrusions of trophoblast cells was observed regardless of the type and developmental stage of the embryos. The results show that a decrease in the number of embryonic cells affects the formation of the primitive endoderm and the development to the egg cylinder in vitro.

In vitro culture of bovine one-cell embryos derived from in vitro fertilization using a semi-chemically defined medium.

The effects of alteration of metabolic substrates and supplementation of amino acids on in vitro development of bovine embryos were examined. One-cell embryos derived from in vitro fertilization were cultured using a semi-chemically defined medium, Brinster's Medium, containing 3 mg/ml bovine serum albumin (BMOC-3) as a basal medium. BMOC-3 did not support embryonic development to the morula stage, but blastocysts were obtained by omitting glucose and lowering the lactate level from 20.12 to 3.0 mM and the pyruvate level from 0.5 to 0.3 mM (mBMOC-3). Supplementation of amino acids (glutamine plus the other 12 essential amino acids, as well as further addition of 7 non-essential amino acids) improved the embryonic developmental rate to the blastocyst stage with an increase in cell number equal to that for co-cultured embryos with bovine oviductal cells in TCM 199 with 10% fetal calf serum. These results clarified that the one-cell bovine embryo can develop to the blastocyst stage in a semi-chemically defined medium, and that amino acids support embryonic development in vitro.

Developmental competence of oocytes from the ovaries of repeat-breeding cows after in vitro fertilization.

The present study examined the developmental competence of oocytes from the ovaries of repeat-breeding cows after in vitro fertilization (IVF). The number of oocytes recovered from eight repeat-breeders averaged 45. Following IVF, the average number of blastocysts obtained was 5 (range from 1 to 7) per cow. Twenty-two frozen-thawed blastocysts were transferred to recipient animals and produced nine pregnancies (41%). Eight normal calves were subsequently born.

Stimulatory effects of insulin on the development of bovine embryos fertilized in vitro.

To obtain information about the effects of insulin on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol (PVA). The percentage of embryos reaching the morula stage was not affected by the addition of 5 micrograms/ml insulin alone, but insulin showed a beneficial synergistic effect with amino acids (AAs) on the embryonic development to morula. Supplementation of insulin (1-100 micrograms/ml) with mSOF+AAs did not improve the percentage of embryos developing to the blastocyst stage. However, a significant increase in cell numbers of blastocysts was observed with the addition of 1 mM glucose and 1 or 10 micrograms/ml insulin from the morula stage. These results show that insulin has a beneficial effect on the preimplantation development of bovine embryos in the presence of AAs and/or glucose, and suggest that insulin improves embryonic development by stimulating AAs transport and/or glucose uptake.