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.

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.

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.

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.

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos.

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

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.

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.

Differentiation of closely related species by DNA hybridization.

The specificity of genomic DNA probes for species differentiation by slot blot hybridization has been investigated. Experiments have been performed investigating species differentiation between monkey and human and between cattle, goat and sheep. It is demonstrated that cross hybridization between probe and DNA sequences from closely related species is reduced by addition of unlabelled DNA from the cross hybridizing species. Quantitative species differentiation is shown possible for all species although with different detection limits. For differentiation between cattle and sheep or goat the detection limits are determined to less than 0·01% whereas the detection limits for differentiation between the closely related species sheep and goat are about 10%.

Species differentiation of heated meat products by DNA hybridization.

A method for quantitation of pork in heat-treated meat products is described. The procedure involves isolation of DNA from meat samples followed by a determination of the average size of DNA fragments by agarose gel electrophoresis. The DNA is then immobilized on nylon membranes and hybridized with a (32)P-labelled probe made from genomic porcine DNA. The signal intensity from filter-bound DNA probe is determined by laser densitometry of the autoradiographs. Functional relationships between the signal intensity and the fragment size of the DNA as well as signal intensity and the amount of pork DNA have been established. Pork DNA can therefore be quantified using DNA standards with the same fragment size as the actual sample DNA. Samples of known composition and heat treatment have been investigated. The detection limit has been determined to approximately 0·1% pork in beef whereas for heat-treated samples the detection limit has been determined to approximately 0·5% pork in beef. Examples are given in which the present method has been applied on commercial canned and cold-stored foods. The results of the investigation indicate that the DNA-hybridization technique applying (32)P-labelled probes can be used for quantitative determinations in quality control of heat-treated meat products.

A comparison of DNA-hybridization, immunodiffusion, countercurrent immunoelectrophoresis and isoelectric focusing for detecting the admixture of pork to beef.

Using DNA-hybridization at least 0·5% raw pork admixtured to beef could be detected using total genomic pig DNA as well as a cloned pig-specific DNA fragment as a DNA probe. Although signal intensity increased with increasing amounts of pig-DNA, a precise quantitation of pork in samples was not possible. Compared to this the sensitivity for detecting raw pork in beef found by Countercurrent Immunoelectrophoresis was 0·4%; by Immunodiffusion, 1·1%, and by Isoelectric Focusing, 5·0%.

Bovine leukocyte adhesion deficiency in Danish Holstein-Friesian cattle. I. PCR screening and allele frequency estimation.

A screening program for bovine leukocyte adhesion deficiency (BLAD) in Danish Holstein-Friesian cattle has been initiated. During the first months 1611 animals were tested by a PCR based assay. Of these animals 1256, 346, and 8 were assigned normal, BLAD carriers, and BLAD affected animals, respectively. One bull, born as a co-twin, showed weak reaction for the BLAD allele on DNA isolated from leukocytes, but a normal genotype on DNA isolated from semen. Chromosome analysis showed that this bull was a blood chimaera. Estimation of the BLAD allele frequency upon the PCR test results showed that around 450 Danish calves born in 1991 might have been affected with the recessive disorder.

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.