A tortoiseshell male cat: chromosome analysis and histologic examination of the testis.

Tortoiseshell coat color is normally restricted to female cats due to X-linkage of the gene that encodes the orange coat color. Tortoiseshell male cats do, however, occur at a low frequency among tortoiseshell cats because of chromosome aberrations similar to the Klinefelter syndrome in man: the extra X chromosome of a 39,XXY karyotype introduces the possibility of an orange and a non-orange allele which produce the mixture of orange and non-orange coat spotting known as tortoiseshell. We analyzed the chromosome complement of a fibroblast culture and did histological examinations of testicular tissue from a tortoiseshell male cat referred to us. Chromosome analysis using RBA-banding consistently revealed a 39,XXY karyotype. Histological examinations of testis biopsies from this cat showed degeneration of the tubules, hyperplasia of the interstitial tissue, and complete loss of germ cells. Immunostaining using anti-vimentin and anti-VASA (DDX4) showed that only Sertoli cells and no germ cells were observed in the testicular tubules. As no sign of spermatogenesis was detected, we conclude that this is a classic case of a sterile, male tortoiseshell cat with a 39,XXY chromosome complement.

Synovial folds in equine articular process joints.

REASONS FOR PERFORMING STUDY: Cervical synovial folds have been suggested as a potential cause of neck pain in humans. Little is known about the extent and characteristics of cervical synovial folds in horses. OBJECTIVES: The objective of this explorative study was to determine the frequency of synovial folds in equine cervical articular process joints and to provide a characterisation of the size and morphology of the synovial folds. METHODS: Equine cervical articular process joints from 6 horses were included in the study, ranging from cervical vertebra 2 (C2) to cervical vertebra 7 (C7) bilaterally. The articular process joints were dissected, and the cranial and caudal synovial folds of each joint were measured and embedded in paraffin. Synovial folds were analysed histologically and classified according to type, as adipose, fibrous and mixed type. Factors potentially influencing fold size were investigated, including joint number (from C2/C3 to C6/C7), fold type, position of fold within the joint (cranial or caudal) and side of neck (right or left). RESULTS: Synovial folds were identified in 98% of cervical articular process joints examined. The width of the synovial folds varied from 4 to 41 mm, and the height from 1 to 17.8 mm. Thirty-eight per cent of the synovial folds were of adipose type, 41% of fibrous type and 21% of mixed type. Synovial fold size was significantly influenced by the side of the neck and fold type. CONCLUSIONS AND POTENTIAL RELEVANCE: This study provides a characterisation of the frequency, size and morphology of equine cervical synovial folds in 6 horses. Synovial folds were present in 98% of the cervical articular process joints examined, and the size of the synovial folds indicates that they could be damaged by acute injury or chronic disease in the cervical articular process joints.

Foetal life protein restriction in male mink (Neovison vison) kits lowers post-weaning protein oxidation and the relative abundance of hepatic fructose-1,6-bisphosphatase mRNA.

Foetal life malnutrition has been studied intensively in a number of animal models. Results show that especially foetal life protein malnutrition can lead to metabolic changes later in life. This might be of particular importance for strict carnivores, for example, cat and mink (Neovison vison) because of their higher protein requirement than in other domestic mammals. This study aimed to investigate the effects of low protein provision during foetal life to male mink kits on their protein metabolism during the early post-weaning period of rapid growth and to investigate whether foetal life protein deficiency affects the response to adequate or deficient protein provision post weaning. Further, we intended to study whether the changes in the gene expression of key enzymes in foetal hepatic tissue caused by maternal protein deficiency were manifested post-weaning. A total of 32 male mink kits born to mothers fed either a low-protein diet (LP), that is, 14% of metabolizable energy (ME) from protein (foetal low - FL), n = 16, or an adequate-protein (AP) diet, that is, 29% of ME from protein (foetal adequate - FA), n = 16) in the last 16.3 ± 1.8 days of pregnancy were used. The FL offspring had lower birth weight and lower relative abundance of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) and pyruvate kinase mRNA in foetal hepatic tissue than FA kits. The mothers were fed a diet containing adequate protein until weaning. At weaning (7 weeks of age), half of the kits from each foetal treatment group were fed an AP diet (32% of ME from protein; n = 8 FA and 8 FL) and the other half were fed a LP diet (18% of ME from protein; n = 8 FA and 8 FL) until 9.5 weeks of age, yielding four treatment groups (i.e. FA-AP, FA-LP, FL-AP and FL-LP). Low protein provision in foetal life lowered the protein oxidation post-weaning compared with the controls (P = 0.006), indicating metabolic flexibility and a better ability to conserve protein. This could not, however, be supported by changes in liver mass because of foetal life experience. A lower relative abundance of Fru-1,6-P2ase mRNA was observed (P < 0.05), being lower in 9.5-week-old FL than in FA kits. It can be concluded that foetal life protein restriction leads to changes in post-weaning protein metabolism through lower protein oxidation of male mink kits.

Meiotic studies in infertile domestic pig-babirusa hybrids.

Mating of a babirusa (Babyrousa babyrussa) boar and a domestic sow (Sus scrofa) resulted in the birth of 5 live domestic pig-babirusa hybrid piglets. Chromosome analysis of one of the surviving males confirmed that they were domestic pig-babirusa hybrids by revealing the presence of a complete haploid set of 19 porcine chromosomes as well as a complete haploid set of 19 babirusa chromosomes in the karyotype. None of the surviving piglets, two males and one female, had shown signs of sexual maturity at age 27 months. Histological examination of gonadal biopsies from the 2 males revealed that both were azoospermatic. Immunostaining revealed SCP3-positive axial elements in the nuclei of primary spermatocytes, indicating that they were progressing through leptotene and zygotene of meiotic prophase. However, the presence of multiple short stretches of axial elements in pachytene nuclei indicated that this phase was blocked, probably due to aberrant chromosome pairing. Histological examination of the ovaries revealed follicular structures, but oocytes within them were generally degenerated. We conclude that both male and female pig-babirusa hybrids were infertile, most likely due to germ cell death resulting from abnormalities of chromosome pairing during meiotic prophase.

Accuracy of ultrasound-guided injections of thoracolumbar articular process joints in horses: a cadaveric study.

REASONS FOR PERFORMING STUDY: Arthrosis of the articular process joints (APJs) in the caudal thoracolumbar region of horses may cause back pain and subsequent reduced performance or lameness. Ultrasound-guided injections of the APJs of the equine back have been described only briefly in the literature. OBJECTIVES: To evaluate factors affecting the accuracy of intra-articular injections of the APJs in the caudal thoracolumbar region. METHODS: One-hundred-and-fifty-four injections with blue dye were performed on APJs including the T14-L6 region in 12 horses subjected to euthanasia for reasons unrelated to back problems. The backs were subsequently dissected to verify the location of the injectate in relation to the APJs. RESULTS: Twenty-seven percent of the injections were found to be intra-articular and a total of 77% found to be within 2 mm of the joint capsule including the intra-articular deposits. Application of a medial approach and 18 gauge needle were significantly associated with an intra-articular injection or deposition close to the joint capsule. Operator, APJ (location) and back number (chronological) did not significantly affect the accuracy of injection. CONCLUSIONS AND POTENTIAL RELEVANCE: Injection of the vertebral APJ in the thoracolumbar region using ultrasound guidance is a reliable method, as most of the injections were either in or within 2 mm of the joint. Based on the findings of this cadaver study, the medial approach is expected to be the most accurate in live horses. Further investigations are required to evaluate the diagnostic and therapeutic potential of this method in clinical practice.

Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA.

An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG/CGG(n) trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X.

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos.

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

Cartilage-derived retinoic acid-sensitive protein in equine synovial fluid from healthy and diseased joints.

REASONS FOR PERFORMING STUDY: More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD-RAP. OBJECTIVES: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD-RAP in synovial fluid from healthy and diseased joints. METHODS: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD-RAP concentrations were measured using a human CD-RAP ELISA. Intra- and interassay imprecision of the assay were evaluated by multiple measurements on pools of equine synovial fluid. Assay inaccuracy was determined by linearity under dilution. RESULTS: The assay showed moderate to large intra- and interassay variation when applied to equine synovial fluid. Equine CD-RAP was detected in synovial fluid from healthy horses ranged at 8.2-52 ng/ml. Repeated arthrocentesis (after injection of isotonic saline), age, joint or gender did not significantly affect CD-RAP concentrations. Twelve hours after intra-articular injection of lipopolysaccharide, concentrations of CD-RAP were significantly lower than after injection of isotonic saline and remained significantly lower until the end of the study at 144 h. CONCLUSION AND POTENTIAL RELEVANCE: The assay is suitable for longitudinal monitoring of CD-RAP concentration in individual horses. Disease significantly influenced CD-RAP levels. Similar to previous results obtained in man, CD-RAP seems to be a marker of cartilage synthesis and/or regeneration in horses.

Transcription levels of endothelin-1 and endothelin receptors are associated with age and leaflet location in porcine mitral valves.

The aim of the study was to investigate the expression levels of endothelin-1 (ET-1) and ET(A) and ET(B) receptors (ET(A)-R and ET(B)-R) in porcine mitral valves and associate the transcription levels to age, leaflet location and deposition of mucopolysaccharides (MPS). Tissue samples from the chordal and inter-chordal insertion area of the anterior mitral valve leaflet from 11 sows (> or = 2 years of age) and 10 slaughter pigs (approximately 6 months old) were obtained and the relative gene expression levels of ET-1, ET(A)-R and ET(B)-R measured by semi-quantitative real-time PCR. A separate tissue sample was taken for histopathological grading of MPS deposition. The transcription levels of ET-1 (P < 0.0001) and ET(A)-R (P < 0.0004) were significantly higher in leaflets from the sows compared with slaughter pigs. The gene expression of ET(B)-R was not associated to age (P = 0.38), but increased in chordal insertion areas compared with inter-chordal areas (P = 0.01). The expression of ET-1 and ET(A)-R mRNA did not differ significantly between the two leaflet locations. The valve leaflets from sows had a significantly increased degree of MPS deposition compared with slaughter pigs upon histological examination (P = 0.04). In conclusion, an age-related valvular degeneration is observed in porcine mitral valve leaflets and ET-1 is suggested to be involved through action of both ET(A) and ET(B) receptors.

Increased expression of endothelin B receptor in static stretch exposed porcine mitral valve leaflets.

The aim of this study was to evaluate the effect of mechanical stretch on the expression of ET-1 and ET(A)- and ET(B)-receptors in porcine mitral valve leaflets. Leaflet segments from 10 porcine mitral valves were exposed to a static stretch load of 1.5 N for 3.5h in buffer at 37 degrees C together with matching control segments. Subsequently, the mRNA expression of ET-1, ET(A)-R and ET(B)-R was measured by real-time RT-PCR in the chordal insertion areas. The analyses showed an increased transcription of ET(B)-receptors in stretch-exposed leaflet segments compared to unstretched segments median 2.23 (quartiles 1.37 and 2.70) vs. median 1.56 (quartiles 1.38 and 2.17, P=0.03) whereas the mRNA expression of ET(A)-receptors (P=0.90) and ET-1 (P=0.51) remained unchanged. Stretch increased the expression of ET(B)-receptors in porcine mitral valve leaflets. The finding could lead to a better understanding of the pathogenesis of myxomatous mitral valve disease.

Effect of time of artificial insemination on embryo sex ratio in dairy cattle.

The objective of the present study was to examine whether different intervals between insemination and ovulation have an influence on the sex of seven-day-old embryos in dairy cattle. Cows were inseminated once with semen of one of two bulls of proven fertility between 36 h before ovulation and 12 h after ovulation. Time of ovulation was assessed by ultrasound at 4-h intervals. In total, 64 embryos were determined to be male or female. Of these 64 embryos, 51.6% were female. The sex ratio in the various insemination-ovulation intervals (early: between 36 and 20 h before ovulation; intermediate: between 20 and 8 h before ovulation; late: between 8 h before and 12 h after ovulation) did not significantly differ from the expected 1:1 sex ratio (50, 50 and 55% females, respectively). Bull (Bull A and B) and Parity (primiparous and multiparous) had no influence on the expected 1:1 sex ratio either. The number of cell cycles was similar for male and female (P = 0.23) embryos when quality of the embryo (P < 0.0001) was included in the model. The results of this study indicate that, in cattle, the interval between insemination and ovulation does not influence the sex ratio of seven-day-old embryos.

Spontaneous fusion between cancer cells and endothelial cells.

Endothelial cells line the inside of blood and lymphatic vessels, and cancer cells must cross this barrier, first to gain access to the circulation, and, second, to exit and metastasize. How this occurs is incompletely understood. We now demonstrate that human cancer cells are able to fuse with endothelial cells to form hybrid cells displaying proteins and chromosomal markers characteristic of both parent cells. The hybrid cells are viable and capable of undergoing mitosis. Fusions between cancer cells and endothelial cells were shown to occur both in vitro, in co-cultures of human breast cancer cells and endothelial cells, and in vivo, following intravascular dissemination of human breast cancer cells in nude mice. These observations demonstrate a new type of cancer-endothelial cell interaction that may be of fundamental importance to the process of metastasis.

Ultrasonography of the equine cervical region: a descriptive study in eight horses.

REASONS FOR PERFORMING STUDY: In equine patients, the cause of clinical signs possibly related to the cervical region is often difficult to diagnose. Ultrasonography allows quick and noninvasive visualisation, but reference material of the normal equine neck is needed. OBJECTIVES: To describe and document the normal ultrasonographic appearance of transverse scans in the cervical region with emphasis on the synovial articular facet joints, cervical vertebrae and paravertebral structures; and further, to provide images of frozen cross-sections for anatomical reference. METHODS: A study describing the normal ultrasonographic appearance of the cervical anatomy was performed. Transverse scans were obtained from second cervical vertebra (C2) to first thoracic vertebra (T1). Post mortem photographs of frozen cross-sections were obtained as anatomical reference. RESULTS: The structures were clearly visualised by ultrasonography and consistency was found between ultrasonographic images and corresponding cross-sectional anatomy. The articular facets varied between horses and facets (C2 to T1). Discrepancy in the existing anatomical descriptions was found. CONCLUSIONS AND POTENTIAL RELEVANCE: The anatomical and ultrasonographic description provides a reference for ultrasonographic evaluation of equine cervical facet joints, vertebrae and paravertebral structures. The findings and variations found are considered to reflect the naturally occurring variations in horses.

Accuracy of ultrasound-guided intra-articular injection of cervical facet joints in horses: a cadaveric study.

REASONS FOR PERFORMING STUDY: Intra-articular facet joint injection is an established diagnostic procedure in human medicine but there are no reports on its reliability in equine practice. OBJECTIVES: To investigate the accuracy of ultrasound-guided intra-articular injections of the cervical facet joints and to estimate factors influencing the accuracy. METHODS: Sixty injections with blue dye were performed on the facet joints between 2nd and 7th cervical vertebra (C2-C7) on horses subjected to euthanasia for nonorthopaedic reasons. The facet joints were subsequently dissected to verify accuracy of deposition. RESULTS: Seventy-two percent of the injections were found to be intra-articular, 17% were intracapsular and a total of 98% were within 1 mm of the joint capsule. There was a marked effect of gained experience (P < 0.01), but not of other factors tested. CONCLUSIONS AND POTENTIAL RELEVANCE: The results of the present study do not translate directly to injections performed in live horses, but they indicate that the method can be applied as a diagnostic as well as therapeutic procedure in C2 to C7 and that is advisable to practise injections on cadaver specimens before applying the technique.

Morphological assessment of preimplantation embryo quality in cattle.

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.

Cloning and characterization of the bovine EGR4 gene and evaluation as candidate gene for bovine spinal dysmyelination.

Genes of the early growth response (EGR) family encode transcription factors with a highly conserved DNA binding zinc finger domain, which regulate a variety of genes, e.g. late myelin genes. Here, the cloning, genomic structure and expression of the bovine orthologue of the EGR4 gene are reported. The gene consists of two exons and encodes a 482 amino acid protein with a Cys2His2 zinc finger structure. The predicted protein shares between 80 and 87% identity to mouse, rat and human EGR4 proteins and all four species share almost complete identity in the DNA-binding domain. The bovine transcript is alternatively spliced by retaining intronic sequence, giving rise to two different mRNAs differing in three nucleotides and resulting in an extra alanine residue in the longer variant of the predicted protein. The gene was mapped by radiation hybrid (RH) mapping to markers on bovine chromosome 11. EGR4 transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the frontal cortex and cerebellum, and a low expression level was also detected in the liver. The EGR4 gene was evaluated as a candidate gene for bovine spinal dysmyelination (BSD). Sequencing of the gene from a homozygous affected animal and a heterozygous carrier revealed a single base mutation that leads to an amino acid substitution at residue 322 in EGR4. Genotype analysis of this polymorphism in a pedigree segregating for BSD, as well as in a panel of different cattle breeds, and sequence analysis of the entire coding region suggested that the EGR4 is not the gene responsible for BSD. Furthermore, 87 animals of different cattle breeds were screened for single-nucleotide polymorphisms (SNPs) resulting in the identification of two SNPs in EGR4.

Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos.

In current in vitro production (IVP) systems, oocytes lack in vivo dominant and preovulatory follicular development, which may compromise pregnancy and viability of calves born. When an oocyte sets off in vivo on the road toward fertilization, it contains numerous transcripts and proteins necessary to survive the first few cell cycles of embryonic development. It is not yet known during which period of development the oocyte builds up the store, possibly primarily during the major growth phase of the oocyte, which is completed at the time a follicle reaches the size of 3 mm. Here, we investigated to what extent the later phases of follicular development, such as prematuration in the dominant follicle before the LH surge and ensuing final maturation in the preovulatory follicle, contribute to oocyte competence and development into viable biastocysts. Recent studies on in vivo vs in vitro oocyte maturation employed oocytes from an identical preovulatory development by applying ovum pick-up (OPU) twice (before and 24 h after the LH surge) in each cow treated for superovulation with a controlled LH surge. The embryo recovery rates at Day 7 of IVC after IVF were similar: 44% (97/219) for in vivo- vs 41% (87/213) for in vitro-matured oocytes, which shows that the natural environment during final maturation is not essential for the mere in vitro development of the prematured oocyte beyond the 8- to 16-cell stage. However, in vivo maturation appeared to contribute to the oocyte's quality in a more subtle way, as indicated by a significant increase in the proportion of expanded blastocysts and a more physiological degree of chromosome aberrations of the embryos. In blastocysts derived from in vivo-matured oocytes, 21% of the embryos were mixoploid vs 50% from in vitro-matured oocytes, concomitant with a higher number of cells (96 vs 54 per normal blastocyst). The expression pattern of a set of six developmentally important genes was, however, not significantly altered in blastocysts derived from in vivo-matured oocytes. Certain deviations were observed compared with the levels of entirely in vivo-developed control blastocysts, which suggests that the beneficial effects of in vivo maturation are possibly exerted at initial stages of embryonic development. Prematuration in vivo, occurring in a dominant follicle developing from about 8 mm into the preovulatory follicle, is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC). Presumably, the differentially expressed proteins are involved in equipping the oocyte with further developmental competence. Although we have unraveled some important biochemical and cellular biological features of the oocyte, further research on in vivo processes is essential to improve in vitro embryo production in practice.

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis.

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos.

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Chromosomal abnormalities and developmental kinetics in in vivo-developed cattle embryos at days 2 to 5 after ovulation.

The frequency of chromosome abnormalities was investigated in cattle embryos (n = 256) derived from superovulated heifers (n = 35) on Days 2, 3, 4, and 5 postovulation (PO). Interphase nuclei (n = 4358) were analyzed for chromosome abnormalities using fluorescent in situ hybridization with chromosome 6- and chromosome 7-specific probes and the developmental rate was described by scoring cell numbers. We found that 93%, 85%, 84%, and 69% of the embryos from Days 2, 3, 4, and 5 PO, respectively, displayed a normal diploid chromosome number in all cells. Of the embryos containing abnormal cells, mixoploidy was significantly more frequent than polyploidy. The percentage of mixoploidy at Days 2, 3, 4, and 5 PO was 5%, 13%, 16%, and 31%, respectively, whereas the percentages of polyploidy were 2%, 2%, 0%, and 0%, respectively. The mean number of cells per embryo was 4.7, 8, 11.5, and 48.3, respectively, at Days 2, 3, 4, and 5 PO. Thus, in vivo-developed embryos were significantly more advanced than the in vitro-produced (IVP) embryos except for Day 2. In conclusion, a significantly lower frequency of chromosomally abnormal embryos, in particular displaying polyploidy early after fertilization, was seen in in vivo versus IVP embryos, and these chromosomal abnormalities may be inherent to the process of IVP in cattle.