Ovine-specific Y-chromosome RAPD–SCAR marker for embryo sexing

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker ( UcdO43 ). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA. The assay was also confirmed to be specific for the ovine Y-chromosome as bovine, caprine, porcine, murine, and human DNA did not amplify. The ovine embryo sexing method is a duplex PCR system that also includes ZFY/ZFX primers. ZFY/ZFX provide an internal positive control for amplification as well as a means to confirm the results obtained with the UcdO43 primers. All embryo sexing results (36/36) from our method were in agreement with the ZFY/ZFX assay results. However, while our method requires an internal control to detect PCR failure, it has the advantages of not requiring nested PCR or restriction endonuclease digestion of the PCR product, and concerns about cross-species contamination are eliminated.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep

Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.     

Including endophenotypes as covariates in variance component heritability and linkage analysis

Background

Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous.

Results

A novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus ) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified.

Conclusion

PCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.     

The development of PCR methodology for the identification of species of the tapeworm Moniezia from cattle, goats and sheep in central Vietnam

The aims of this study were to investigate the prevalence of Moniezia spp. in domestic ruminants in central Vietnam and to develop a polymerase chain reaction (PCR) technique to distinguish M. expansa from M. benedeni. Among 2040 examined domestic animals (540 cattle, 800 goats, 700 sheep) Moniezia was recovered from 5.4% of cattle, 16.4% of sheep and 20.6% of goats. A set of primers for PCR was designed to classify M. expansa and M. benedeni based on the amplification of DNA corresponding to the internal transcribed spacer of 5.8S rRNA. The 457 specimens (75 from cattle, 162 from goats, 150 from sheep, 30 from horses, 30 from chickens and 10 from dogs) were subjected to PCR for classification of Moniezia spp. PCR products with the expected sizes were amplified from bovine, ovine and caprine specimens. No specific PCR products were found for specimens from horses, chickens and dogs. Of the 75 specimens from cattle, nine were classified as M. expansa and 66 were M. benedeni. Among 162 caprine specimens, 138 were M. expansa and 24 were M. benedeni. The distribution of M. expansa and M. benedeni in 150 ovine specimens was 132 and 18, respectively. These results show that M. expansa is dominant in goats and sheep, whereas M. benedeni is more common in cattle; PCR can be used for classification of these two species.     

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.     

Sex determination in goat by amplification of the HMG box using duplex PCR

The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.     

A molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.     

MAF45, a highly polymorphic marker for the pseudoautosomal region of the sheep genome, is not linked to the FecXI (Inverdale) gene.

A highly polymorphic dinucleotide repeat, or microsatellite, that shows partial sex-linked inheritance in sheep has been isolated from the sheep genome. Our data indicate that the locus is in the pseudoautosomal region approximately 13 cm from the boundary with the sex-linked regions. The locus, designated MAF45, has 12 alleles with a PIC of 0.84. The same primers amplify a single polymorphic locus in cattle and goats. This locus was not linked to the Inverdale gene, an X-linked gene that increases the ovulation rate in sheep.     

Sexual size dimorphism in domestic goats,sheep, and their wild relatives

Sexual size dimorphism (SSD) is a widespread phenomenon in different animal taxa, including the subfamily of goats and sheep (Caprinae), which belongs to the most dimorphic mammalian groups. Rensch's rule describes the pattern of SSD, claiming that larger species generally exhibits higher male to female body size ratio. Agreement with Rensch's rule is manifested by slope of the allometric relationship between male and female body size exceeding one. To test this rule, we analysed the data available in the literature on adult body mass of males and females in domestic goat and sheep breeds (169 and 303, respectively) and 37 wild species/subspecies of the subfamily Caprinae. According to the current phylogenetical hypotheses, there are six distinct monophyletic groups with different levels of SSD (expressed as M/F): (1) wild goats (1.83); (2) wild sheep (1.67); (3) non‐European chamoises, including Ovibos moschatus (1.18); (4) European chamoises (1.27); (5) Budorcas taxicolor (1.01); and (6) Pantholops hodgsonii (1.65). Domestication has led to a remarkable decline in SSD of both domestic goats (1.36) and sheep (1.41). The highest regression slope of the relationship between male and female body size is that estimated for wild goats (1.32), followed by wild sheep (1.24), non‐European chamoises (1.14), domestic sheep (1.13), and domestic goats (1.10). Nevertheless, only the last two values are statistically different from one and thus corroborate Rensch's rule. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 872–883.     

The intake and digestion of a range of temperate forages by sheep and fibre-producing goats

The voluntary intake, digestibility and mean retention time of six temperate forages differing in their chemical composition by 12 adult castrated male Scottish blackface sheep, aged 15 months, and fibre-producing castrated male goats, aged 27 months, and of similar live weight, 40 kg, were described. The creation of a range of chemical compositions was effected through the use of barley straw, and a low- and high-digestibility hay, and the use of ammonia treatment of these forages. A wide range of voluntary intakes (42–78 g DM/kg W^0.75/day), digestibility of dry matter (0.46–0.60) and mean retention times of undigested residues (36–72 h) was achieved through feeding the six forages. Across all the forages fibre-producing goats had higher voluntary intakes, expressed on a metabolic live weight basis, and lower digestibility values than sheep, whereas the mean retention time of the undigested residues was similar for the two species. Within forages goats selected a diet of potentially higher nutritive value, as predicted from chemical composition, with a smaller particle size than sheep. It was concluded that the differences in intake and digestion of temperate forages between sheep and fibre-producing goats are broadly similar to those observed in other experiments between sheep and goats ingesting tropical forages.     

Activities of glutathione-S-transferase and ethoxycoumarin-O-deethylase in tissues of camels, sheep, goats and rats.

1. The activities of the drug metabolizing enzymes ethoxycoumarin-O-deethylase, glutathione-S-transferase, and protein concentrations were measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver than in the kidney and duodenal mucosa in the four species studied. 3. The activities of ethoxycoumarin-O-deethylase and glutathione-S-transferase in liver of male kids were about one third and half of that in adult male goats, respectively. In the kidney and duodenal mucosa of male kids, the activity of glutathione-S-transferase was about 70% and 53% of that in the mature male goat, respectively. In the latter tissues, however, there was no detectable activity of ethoxycoumarin-O-deethylase. 4. In general, goats and sheep had similar activities of the two enzymes which were significantly higher than those found in camels and rats. 5. Some sex-related differences were noted in the activity of the two enzymes studied. Female sheep had significantly higher hepatic glutathione-S-transferase than the male: while the enzyme activity in the kidney and duodenal mucosa of male goats was significantly higher than in females. Male rats had higher hepatic ethoxycoumarin-O-deethylase activity than females.     

Evolutionary history of the genus Capra (Mammalia, Artiodactyla): discordance between mitochondrial DNA and Y-chromosome phylogenies

The systematics of the genus Capra remain controversial in spite of studies conducted using morphology, mtDNA, and allozymes. Here, we assess the evolutionary history of Capra (i) using phylogenetic analysis of two nuclear genes located on the Y-chromosome and (ii) previously published and new cytochrome b sequences. For the Y-chromosome phylogeny, we sequenced segments from the amelogenin (AMELY) and zinc finger (ZFY) genes from all of the eight wild taxa and from domestic goats (Capra hircus). Phylogenetic analysis of the Y-chromosome data revealed two well-defined clades. The domestic goat (C. hircus), the bezoar (Capra aegagrus), and the markhor (C. falconeri) belong to one clade (ML bootstrap value [BP]: 98%), suggesting that domestic goats originated from one or both of these wild species. The second clade (ML BP: 92%) is comprised of all the other wild species. Horn morphology is generally concordant with the Y-chromosome phylogeny. The mtDNA data also revealed two well-defined clades. However, the species in each clade are different from those inferred from the Y-chromosome data. To explain the discordance between Y-chromosome and mtDNA phylogenies, several hypotheses are considered. We suggest that a plausible scenario involves mtDNA introgression between ancestral taxa before the relatively recent colonization of Western Europe, the Caucasus Mountains, and East Africa by Capra populations.     

Genetic aspects of arterial hypertension: the role of Y chromosome and mitochondrial DNA

A study on hybrids from reciprocal crossing of the SHR-SP and the WKY has shown that Y-chromosome and mitochondrial DNA the affect development of the spontaneous hypertension. The Y-chromosome takes part in disorders of baroreceptive sensitivity in phenylnephrine assay associated with hypertension. Although our findings suggest that structural remodelling of peripheral vascular resistance and an increase in noradrenaline-dependent vasocostriction is genetically determined in hypertensive rats, we could not corroborate the role of the Y-chromosome and mitochondrial DNA in the process. A difference was shown between male and female SHR-SP in the level of arterial pressure and in development of the vascular structure changes.     

Arbovirological survey in Silica plateau area, Roznava District, Czechoslovakia

The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against Lipovník (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to Lipovník and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.     

Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.     

Food overlap between Mongolian gazelles and livestock in Omnogobi,southern Mongolia

Botanical and particle size compositions of the feces of sympatric Mongolian gazelles, sheep/goats, and horses collected in southern Mongolia in autumn 2002 were analyzed. The botanical composition of Mongolian gazelles was similar to that of sheep/goats where dicotyledonous plants (64.6% for Mongolian gazelles, 65.6% for sheep/goats), particularly woody fibers were important (39.5% for Mongolian gazelles, 19.5% for sheep/goats). In contrast, horse feces were exclusively composed of graminoids (93.2%). Consequently, food overlap was great between Mongolian gazelles and sheep/goats (Piankas index: 0.977) but was small between Mongolian gazelles and horses (0.437) and sheep/goats and horses (0.421). Particle size distributions were also similar between Mongolian gazelles and sheep/goats, whereas they were different between horses and Mongolian gazelles and horses and sheep/goats. These results support our expectation based on the Jarman-Bell principle. Although interspecific competition cannot be inferred from a mere food overlap, our analyses suggest that sheep and goats are potential competitors for Mongolian gazelles. Therefore, the increase in the numbers of domestic sheep/goats might pose a risk for Mongolian gazelle populations.     

Serological Evidence of Rift Valley Fever Virus Circulation in Sheep and Goats in Zambézia Province,Mozambique

Rift Valley fever (RVF) is endemic in most parts of Africa and has also been reported to occur in the Arabian Peninsula. It is responsible for significant morbidity and mortality, particularly in livestock, but also in humans. During the last two decades several outbreaks of RVF have been reported in countries in Southern Africa. In contrast to other countries, no clinical disease has been reported in Mozambique during this period. In a serological study conducted in 2007 in five districts of Zambézia Province, Mozambique, of a total of 654 small ruminants sampled (277 sheep and 377 goats), 35.8% of sheep sera and 21.2% of goat sera were positive for RVF virus (RVFV) antibodies in a virus neutralization test (VN) and in an IgG enzyme-linked immunosorbent assay (ELISA). In 2010, a cross-sectional survey was conducted in 313 sheep and 449 goats in two districts of the same province. This study revealed an overall seropositivity rate of 9.2% in sheep and 11.6% in goat and an increased likelihood of being seropositive in older animals (OR = 7.3; p<0.001) using an IgG ELISA. 29 out of 240 animals assessed for RVF specific IgM by ELISA were positive, suggesting recent exposure to RVFV. However, a longitudinal study carried out between September 2010 and April 2011 in a cohort of 125 of these animals (74 sheep and 51 goats) failed to demonstrate seroconversion. The results of the study indicate that RVFV circulates sub-clinically in domestic small ruminants in Zambézia Province.     

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.     

Supraependymal cells occurrence in ventricular system of small ruminants. Scanning electron microscopy study

The third cerebral ventricle ependymal lining including eminentia medians was studied by means of SEM in 20 sheep, 13 goats and one goat hermaphrodite. Supraependymal cells in addition to the usual supraependymal structures were observed. In sheep, they occurred in the infundibular low part only in females during oestrus. In goats, they were present in almost every case with the exception of male animals during the "rest" period (April). The number, topography and-to some extent--the appearance of the goat supraependymal cells were in relation to the animal's sex, and the females to the ovarian cycle phase. The supraependymal cells were found on the eminentia mediana surface only in the goats. In small ruminants, the processes of most supraependymal cells formed the ruffled membranes and only the eminentia mediana supraependymal cells--and in hermaphrodite also in the infundibular rostral part--resembled more neurons.