A cytogenetic study of breeding boars in Canada.

Chromosome abnormalities are well known for their negative impact on the reproductive performance of carriers. Such abnormalities could have severe effect on animal industries which rely heavily on efficient reproduction. We conducted a cytogenetic survey of breeder pigs from 4 different Canadian farms to investigate the frequency of chromosome abnormalities and to assess their reproductive impact on pig populations. Our study revealed that 50% of the 'hypoprolific' boars and 2.5% of the young boars raised for service in artificial insemination were carriers of chromosome anomalies while no chromosome defect was noted in any of the 'proven' breeder boars. G-banding technique to determine the type of abnormalities detected 3 previously unreported translocations involving chromosomes 1 and 6, chromosomes 10 and 13 and chromosomes 9 and 14. The reciprocal nature of these translocations was confirmed either using fluorescent in situ hybridization (FISH) technique or immunostaining for synaptonemal complex delineation and were named rcp(1;6)(p22,q12), rcp(10;13), and rcp(9;14) (p24;q27), respectively. Prolificacy of 1/6 and 10/13 translocation carriers was noted to be reduced by more than 40% compared to their normal counterparts while it was reduced by 26% in carriers of the 9/14 translocation. Carriers of 1/6 and 9/14 translocations displayed a higher repeat breeding tendency, compared to their herd average (5 and 16%, respectively). While for the 9/14 translocation the prevalence of stillbirths was lower than that in their herd [8.7 vs. 10.4% (p < 0.001)]. The present results, albeit based on a relatively small number of pigs, indicate that the prevalence of chromosome abnormalities could be much higher in Canadian pigs compared to that reported in European pigs and underline the urgent need to initiate cytogenetic screening programs as one of the effective ways to reduce reproductive problems in Canadian pig populations.

Veterinary cytogenetics: past and perspective.

Cytogenetics was conceived in the late 1800s and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance of chromosomes to human health and disease was realized more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation. Veterinary cytogenetics has benefited from the information generated in human cytogenetics which, in turn, owes its theoretical and technical advancement to data gathered from plants, insects and laboratory mammals. The scope of this science has moved from the structure and number of chromosomes to molecular cytogenetics for use in research or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide polymorphism array-based karyotyping and automated systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well exploited mainly because of the cost-driven nature of demand on diagnosis and treatment which often out-weigh emotional and sentimental attachments. An area where the potential of veterinary cytogenetics will be fully exploited is reproduction since an inherited aberration that impacts on reproductive efficiency can compromise the success achieved over the years in animal breeding. It is gratifying to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization using custom-made microarray platforms that allow the construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications. Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear that the importance of the achievements to date are bound to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.

Differences in the pattern of X-linked gene expression between fetal bovine muscle and fibroblast cultures derived from the same muscle biopsies.

The sex determination system in mammals creates an imbalance between males and females in the number of X chromosomes. This imbalance is compensated through transcriptional silencing of one of the two X chromosomes in female diploid cells by epigenetic modifications. Although common for mammals, X inactivation shows marked species-specific differences in mechanisms and end results, and provides a unique opportunity to study epigenetic regulation of gene expression. The aim of the present study was to establish the expression pattern of selected X-linked genes in bovine fetal muscle tissue and muscle fibroblast cultures in order to follow possible modifications at the transcriptional level attributable to in vitro culture. We used heterologous cDNA microarray hybridization and quantitative real-time PCR to study the pattern of expression of X-linked genes including SLC25A6, GAB3, MECP2, RPS4X, JARID1C, UBE1, BIRC4 and SLC16A2. Quantitative real-time PCR analysis in fetal bovine muscle showed higher transcript levels in females for all X-linked genes tested with the exception of SLC25A6, with differences being significant for RPS4X, JARID1C and UBE1. The expression in fibroblast cultures derived from the same samples differed, with significantly higher levels for UBE1, GAB3 and BIRC4, while the rest of the panel of X-linked genes remained unchanged. The changed expression pattern in vitro, probably reflecting modifications in the epigenetic mechanisms that regulate transcriptional activity and gene silencing in X inactivation, has important implications for the advancement of new biotechnologies such as somatic cell nuclear transfer and stem cell therapy.

Genetics then and now: breeding the best and biotechnology.

In the past, domesticated animals were genetically improved by identifying meritorious individuals, mating animals displaying desired traits, continued breeding of related animals to perpetuate their superior traits and crossbreeding when inbreeding depression became evident. Today, assisted reproduction and biotechnology allow breeders to design and direct the reproductive course, disseminate desired traits and hasten genetic improvement. Generation interval can be greatly reduced by combining artificial insemination, which is the oldest and most widely used assisted reproductive technology, with the more recent techniques, such as oestrus synchronization, superovulation, ovum pick up from immature females even out of breeding season, and in vitro embryo production and transfer. Furthermore, the sex and genetic make-up of the offspring can be selected by using sex-sorted sperm for insemination, marker-assisted selection, functional deletion or addition of specific genes to the offspring's genome, or somatic cell nuclear transfer for cloning. However, the poor success rates with some of these procedures have delayed their large-scale application which, in turn, has hindered the proper evaluation of their genetic impact. The potential genetic consequences of some of these approaches merit the same degree of diligent evaluation that is currently extended to the procedures used for overcoming their 'technical' inefficiencies.

The fragile X in cattle.

In search of an animal model for the human fragile X syndrome, the chromosomes of Holstein cows were examined. This breed was chosen because of previous studies on the baldy calf syndrome. An achromatic gap was observed at a specific site on the X chromosome closer to the centromere than that identified in humans. This unstained gap was found in 3%-4% of cells of the following four animals: an affected calf, her sister, their mother, and an unrelated Holstein cow. The bovine fragile X may not be analogous to the human fragile X but its location may be important as a genetic marker in linkage studies involving the loci for hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G-6-PD).

Effect of malonaldehyde and acetaldehyde on cultured mammalian cells. Production of micronuclei and chromosomal aberrations.

The genotoxic effects of malonaldehyde (MA) and acetaldehyde (AA) were investigated using primary cultures of rat skin fibroblasts. Exposure to MA at 10(-4) to 10(-3) M concentrations resulted in dose-dependent production of micronuclei. MA was approx. 10 times as potent as AA with respect to micronuclei formation. Treatment with 10(-4) and 10(-3) M concentrations of MA for 12 h produced chromosomal aberrations (chromosomal fragments, achromatic lesions and chromatid breaks) in 14 and 34% of metaphases, respectively. At 24 h the corresponding frequencies were 46 and 52%. AA at analogous concentrations produced aberrations in 4 and 14% of metaphases at 12 h, and 20 and 40% at 24 h. Dose-dependent increases in aneuploidy were seen at 10(-4) M and higher concentrations of both aldehydes, with incidences twice as high for MA as for AA.

X-autosome translocation and low fertility in a family of crossbred cattle.

An investigation was carried out on a family of Limousin-Jersey crossbreds exhibiting low fertility in the females, to determine the impact of a previously identified X-autosome translocation (X-AT) on the reproductive performance of the carrier cows. Three of the identified translocation carriers, including a cow and two of her daughters, were maintained at our University Research Station and artificially inseminated periodically with semen from different bulls of known fertility. Attempts to breed the X-AT carriers resulted in high rates of return to estrus between days 28 and 60, abortions between days 121 and 235 after insemination, and a total of 13 live births including 4 translocation carrier calves. Results of superovulation and embryo retrieval trials on X-AT carriers revealed significantly higher proportions of unfertilized and uncleaved ova and abnormal embryos compared to those from normal cows, and no pregnancy in the recipients transferred with morphologically normal blastocysts from X-AT carriers. While the higher rates of failed fertilization and cleavage, abnormal embryos and return to estrus in X-AT carriers could be attributed to chromosome imbalance expected in their gametes, the relatively high prevalence of abortion (late in gestation) was unexpected. Our observations on the fetuses expelled by X-AT carriers after 5 months of gestation indicated that a majority (three out of four) of these fetuses were products of abnormal (3:1) segregation in meiosis I and that these chromosomally unbalanced (hyperdiploid) conceptuses were able to survive early embryogenesis and fetal life up to the end of the second trimester. We hypothesize that their relatively long in utero life and the absence of any overt birth defects may be attributable to the type of chromosomes over-represented in these fetuses and that their eventual expulsion may have been the result of selection against the clonal population of cells in which the altered X carrying a segment of chromosome 23 (Xp(+)), remained inactive.

Fetal fluid steroids and their relationship to gonadal steroid secretion in single and twin bovine fetuses.

Steroid concentrations in the fetal fluids of 153 single and 69 twin bovine pregnancies, ranging in age from 35 to 125 d of gestation, were studied to compare gonadal steroid secretions in vitro with the concentrations found in amniotic and allantoic fluids during the early stages of sex differentiation. Among the steroids measured in fetal fluids, only the testosterone level showed a correlation with the amount secreted by the gonads. Significantly higher concentrations of testosterone were associated with male fetuses than with female fetuses. The concentrations of androstenedione, estradiol and estrone in both fetal fluid compartments were generally correlated with age, reflecting the extra-gonadal source of steroids in these fluids. Androstenedione levels in fetal fluids were significantly higher in twins than in singletons, suggesting that this parameter may be useful for the diagnosis of fetal sex and/or type of pregnancy.

Pulmonary macrophage mobilization in hamsters after cessation of smoking.

Male Syrian hamsters were exposed to smoke from three different types of cigarettes rated as mild, medium, and strong on the basis of their smoke chemistry for 4 weeks. These hamsters were euthanized at 0, 1 and 2 weeks of recovery after the last smoke exposure to study the pulmonary cell response after cessation of smoking. The free macrophage count determined on lung sections stained with hematoxylin and eosin was used as the index to evaluate the rate of recovery from smoke-related insult caused by the different types of cigarettes. The number of macrophages in the smoke-exposed hamsters was consistently greater throughout the recovery period than in the controls, depending on the amount of particulate components including tar and nicotine in the test cigarettes. The macrophage mobilization peaked at the first week of recovery and then showed a tendency to decrease at the second week. At the second week, however, the hamsters exposed to smoke from strong or medium cigarettes still showed significantly higher counts than the controls, while no significant change was noted in those exposed to smoke from mild cigarettes. These results indicate that the process and duration of recovery after cessation of smoking may depend on the extent of particulate matter impinging on the respiratory tract, so that strong cigarettes would be more hazardous because of greater number and longer retention of macrophages in the lung.

Rapid method for the examination of platelet morphology.

A simple, rapid method is described for preparing platelets of domestic animals for light and electron microscopic examinations. The method involves the isolation of the platelet rich plasma (PRP) by centrifugation of the whole blood for five minutes followed by recentrifugation of PRP in a Wintrobe tube at 4500 rpm for 20 minutes to separate the blood components into four distinct layers composed exclusively of plasma, leucocytes, platelets and erythrocytes. A short fixation (20 minutes) by replacing the plasma layer with glutaraldehyde before breaking the bottom of the tube with a glass cutter allows the cellular contents to be gently pushed out of the tube in the form of a cylindrical semisolid pellet, with a wooden applicator stick. A further three hour fixation of the pellet in fresh glutaraldehyde before cutting out the middle layer for routine processing for light and electron microscopy provides pure preparation of platelets with a minimum of morphological distortion.

Testicular cells in hybrid water buffaloes (Bubalus bubalis).

Meiotic chromosome behaviour and testicular histology were studied in water buffaloes (Bubalus bubalis) including two river (Murrah), two swamp and three F1 (Murrah cross swamp) hybrids aged between two and two and a half years, from testicular biopsies obtained by an open surgical method. Meiotic preparations revealed spermatogonial metaphases, pachytene, diplotene, diakinesis, first and second meiotic metaphases and spermatozoa in all three types of buffalo. Chromosome sets ranging from 22 to 26 (most frequent, 24 and 25) with many cells carrying univalent, bivalent and multivalent configurations were observed in hybrids, whereas the meiotic cells in the Murrah and swamp showed chromosome sets exclusively of 25 and 24 (bivalents) respectively. Histological examination of the hybrid testis revealed a large proportion of degenerating spermatocytes and abnormal spermatids in the process of spermiogenesis suggesting that the various synaptic associations leading to unbalanced gametes may be responsible for the degenerating germ cells in the hybrids. The unbalanced meiotic products will probably lead to selection against such spermatozoa or early embryos after fertilisation. Due to a large percentage of germinal epithelial cells in F1 hybrids being wasted, the fertility of backcross and F2 generations will be subnormal.

Reciprocal translocation (13;20)(q12;q22) in an Icelandic sheep.

Cytogenetic examination of G-banded lymphocyte chromosomes of an Icelandic ram from a line with a history of poor fertility revealed a rcp (13;20) (q12;q22) translocation. Meiotic studies showed a quadrivalent configuration at diakinesis and this was confirmed by C-banding.

Quinolone arthropathy in immature rabbits treated with the fluoroquinolone, PD 117596.

To study the potential of the fluorquinolone, PD 117596 to cause arthropathy in experimental animals, immature rabbits were orally administered the drug for five days at 0, 100, 350, 500 and 750 mg/kg. Characterization of changes induced in major synovial joints was based on: macroscopic and histopathologic observations, transmission electron microscopic examinations and magnetic resonance imaging. Preferentially targeting the knee, PD 117596 produced vesicles and erosions in articular cartilage which resembled, morphologically, those described in other laboratory species. Lesion incidence was not clearly dose-related. In the perivesicular region, degenerate chondrocytes were intermixed with hypertrophic cartilage cells and chondrocyte clusters. Ultrastructurally, hypertrophic chondrocytes were the consequence of karyomegaly and RER proliferation. Matrix density was reduced due to collagen and proteoglycan loss. Joint structures were readily visualized by magnetic resonance imaging which identified thickened articular cartilage, surface irregularities consistent with ruptured vesicles and separation of opposing articular surfaces secondary to synovival effusions. The immature rabbit, although less sensitive than the juvenile dog to the arthropathic effects of quinolones, was nonetheless a good model to study this experimental osteoarticular disease.

An equine cryptorchid with testicular and ovarian tissues.

Cytogenetic and histological studies were carried out on an intersex horse which was diagnosed clinically as a cryptorchid. Surgery confirmed the horse to be a bilateral abdominal cryptorchid and histological examination revealed ovarian tissue associated with the left epididymis. Chromosome analysis of cultured cells from testicular tissue, ovarian tissue and skin revealed 64-XX and 64-XY make-up, the left gonad containing a greater preponderance of XX cells over XY cells. The external characteristics and behaviour of the horse were indistinguishable from that of a "routine" cryptorchid. Other cases of equine intersexes are reviewed and theories for the discrepancies between genetic, gonadal and phenotypic sex are discussed.

Open canalicular system of platelets in porcine stress syndrome.

A study was undertaken to test whether a previously reported alteration in platelet morphology could be of predictive value for the detection of stress-susceptibility in pigs. Platelets from 20 normal pigs, nine pigs classified as stress-susceptible on the basis of their response to halothane challenge, and 11 siblings of halothane reactors belonging to two different breeds were subjected to electron microscopic examination. A quantitative analysis of electron micrographs, based on the extent of dilatation of the open canalicular system in platelets and the percentage of affected platelets, revealed that halothane reactor pigs could be distinguished from normal animals on the basis of their open canalicular system score. The discrete nature of the score categories in siblings indicates that platelet alteration may be an inherent component of the porcine stress syndrome and suggests that some of the false negatives in the halothane test may be identified as stress-susceptible on this criterion. Further studies involving a larger number of halothane reactors and siblings are needed to ascertain the consistency of the open canalicular system features and eventually, to develop a simple test system based on platelet alterations for the detection of stress-susceptibility in pigs.

Steroidogenesis in fetal bovine gonads.

Gonadal steroidogenesis in bovine fetuses of 40 to 125 days gestation was examined using histochemical procedures and radioimmunoassay on gonadal cultures to determine the physiological correlates of gonadal morphogenesis in cattle. Gonadal morphology and the in vitro secretion patterns were distinct between the sexes by 45 days when testes secreted significantly higher levels of testosterone and androstenedione and lower levels of estrone and 17 beta-estradiol that the ovaries (p less than 0.0001). It would appear that the main steroid route in the ovaries of 45 to 70 day old fetuses is the androstenedione to estrone to 17 beta-estradiol pathway. The high estrone secretion and the decreasing levels of 17 beta-estradiol and testosterone in the ovaries of 70 to 125 day fetuses suggest an inhibition of 17 beta-hydroxysteroid dehydrogenase activity. It is postulated that this shift in steroid biosynthetic pathways may be related to the change in cellular events from mitosis to meiosis in fetal ovaries.

The Sertoli cell of the water buffalo--an electron microscopic study.

The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.

Evaluation of a commercial creatine kinase screening test for malignant hyperthermia (porcine stress syndrome).

Two hundred and seven boars entering a Record of Performance Test Station at New Hamburg, Ontario were screened for susceptibility to malignant hyperthermia or the porcine stress syndrome. Screening tests included the determination of whole blood creatine kinase levels by a commercially available test using the method of bioluminescence and a halothane challenge. The validity of the bioluminescent or whole blood creatine kinase test as a screening mechanism for malignant hyperthermia was evaluated in boars in a field trial. The susceptibility of these animals to malignant hyperthermia or the porcine stress syndrome was unknown at the time of the study. It was determined in the initial field trial that 76.3% or 158 of the 207 boars tested by the bioluminescent creatine kinase screening test were stress susceptible. In contrast, only one boar (0.5%) reacted to a standard five minute halothane challenge. After further examination of the commercial screening test, little correlation was found to exist between the bioluminescent and Rosalki methods of creatine kinase determination. The coefficient of analytical variation for the bioluminescent method of creatine kinase determination ranged from 17.6% at a mean of 359 LU to 21.9% at a mean of 318 LU. Similarly, the coefficient for the Rosalki technique ranged from 7.1% at a mean of 324 U/L to 14.0% at a mean of 64 U/L. In addition, little correlation was found to exist between creatine kinase levels as determined by the bioluminescent method and outcome to the halothane or halothane/succinylcholine challenge, age of boars in weeks or percentage gain in weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cigarette smoke on the surface structure of the conjunctival epithelium.

Syrian golden hamsters were exposed to cigarette smoke in an intermittent smoke exposure device, and the surface structure of the tarsal conjunctiva was compared with that of sham-smoked and cage-held controls. Scanning electron microscopy revealed distortion and loss of microvilli of the conjunctival cells in the smoke-exposed animals.

Congenital abnormalities of the goat.

Congenital abnormalities of genetic and environmental causes constitute a striking proportion of the afflictions seen in goats. These include a variety of malformations and metabolic diseases that could occur in all breeds but tend to exhibit predisposition in some breeds of goats. Genetic abnormalities for which the carrier state is detectable with the aid of enzymes and surface protein markers can be eliminated from goat populations, whereas common polygenic disorders including udder problems in does and gynecomastia in bucks are more difficult to eradicate because the mutant genes responsible for these traits generally do not declare themselves until inbreeding brings together a critical concentration of liability genes to create a crisis. A substantial reduction of common abnormalities in this species, such as intersexuality in dairy breeds, abortion in Angora breed, and arthritis in the Pygmy breed, will require a change in breeders' preference and selection practice. In making these changes, however, the beneficial traits will have to be balanced against the undesirable effects of the selected mutant genes (pleiotropy), which hold the key to success or failure of a breed under domestication.