Monitoring of genetic diversity in the endangered Martina Franca donkey population.

The Martina Franca (MF) donkey, an ancient native breed of Apulia, was mostly famous for mule production. The breed was at serious risk of extinction in the 1980s following the decrease in demand for draft animals because they were increasingly replaced by agricultural machinery. Much has been done in the last few decades to safeguard the existing donkey breeds, but the situation remains critical. Successful implementation of conservation measures includes an evaluation of the present degree of breed endangerment, so the aim of this work was to analyze the demographic and genetic parameters of this breed to suggest effective conservation strategies. With a current breed register counting less than 500 recorded animals, the pedigree data set included 1,658 MF donkeys born between 1929 and 2006. Analyses were carried out on the whole data set as well as on a smaller one consisting of 422 living animals. Demographic and genetic variability parameters were evaluated using the ENDOG (v4.6) software. The pedigree completeness level was evaluated as well as the generation length, which was calculated for each of the 4 gametic pathways. This information was obtained from animal birth date records together with those of their fathers and mothers. The effective number of founders (f(e)), the effective number of ancestors (f(a)), the founder genome (f(g)), individual inbreeding (F), average relatedness (AR), and the rate of inbreeding per generation were analyzed to describe the genetic variability of the population. Because pedigree depth and completeness were appropriate, especially regarding the current population, the parameters defining genetic variability, namely, f(e), f(a), f(g), F, and AR, could be reliably estimated. Analysis of these parameters highlighted the endangerment status of the MF donkey. Our special concern was with the increased percentage of males and females exhibiting increased AR values. Moreover, the effective size of the current population, 48.08, is slightly less than the range of the minimum effective size, and the rates of inbreeding per generation found in the current MF population exceed the maximum recommended level of 1%. Such a scenario heightens concerns over the endangered status of the MF breed and calls for proper conservation measures and breeding strategies, such as selecting individuals for mating when relationships are below 12.5%.

Hot topic: Gene duplication at the α-lactalbumin locus: finding the evidence in water buffalo (Bubalus bubalus L.).

Studies on milk proteins revealed that a qualitative and quantitative polymorphism may often be found regarding alpha-lactalbumin (alpha-LA). In mammals, a similar phenomenon was widely documented in the alpha-globin system as the result of a gene duplication. The presence of several differently expressed alpha-lactalbumin gene (LALBA) products suggests that the mechanism underlying this phenomenon may involve nonallelic genes. To check this hypothesis, an experiment was set up to investigate the LALBA gene arrangement of a water buffalo exhibiting an alpha-LA phenotype characterized by a double-band pattern on PAGE isoelectric, focusing analysis of milk protein. In particular, the relative amount of protein inferred from the different intensity of the bands was consistent with a gene duplication. Thus, leukocyte DNA was extracted from a blood sample of the buffalo and amplified with 4 primers (2 RV-IVFW for PCR and 4 FW-IRV for nested PCR). The intergenic segments of the assumed duplicated gene were then amplified with 2 different PCR protocols. First, the segment limited by the third exon in the upstream gene and the second exon in the downstream gene was amplified by simple PCR, which gave aspecific results. Second, this PCR product was subjected to nested PCR, amplifying the segment limited by the fourth exon in the upstream gene and the first exon in the downstream gene, yielding an amplified nucleotide fragment of about 6,200 bp. Blood samples from an additional 15 buffalos were then analyzed in the same manner. The results obtained from the new samples confirmed the presence of an amplified nucleotide fragment of about 6,200 bp in most of them, though they all were characterized by an alpha-LA monomorphic phenotype. A couple of 6,200-bp fragments obtained were purified, cloned in pGEM-T easy vector system (Promega, Madison, WI) and sequenced. The sequence of the large DNA segments, containing the intergenic portion, was aligned with the LALBA gene (accession number AF194373; http://www.ncbi.nlm.nih.gov/Database/index.html). They both were found to coincide with the portion containing exon 4 and the untranslated region at the 3' end of the upstream gene and with the portion containing exon 1 and the untranslated region at the 5' end of the downstream gene. These results confirm the hypothesis that a tandemly repeated copy of the LALBA gene is present in water buffalo.

Relationships among functional markers, management, and husbandry in sheep: a Mediterranean case study.

Most sheep farmers are aware of the importance of monitoring animal health and well-being for profitable sheep production. Unfortunately, there are only a few benchmarked functional measures of sheep well-being but much can be gained from our understanding of other species. Moreover, comprehensive monitoring programs may be complex and relatively expensive to implement. Hence, this work reports the results of a research study on the usefulness of functional markers in measuring dairy sheep well-being, taking into account farm management and environmental conditions. The study was conducted on 11 farms breeding Italian islander sheep breeds. The husbandry and management parameters of each farm were assessed and, based on the findings, the farms were scored in ascending quality order. Flock information concerned housing, milking system, pen size, grazing hours, health management, and stockmanship. Medical history, clinical data, the most relevant haematological, chemical and biochemical parameters, as well as the haemoglobin genotype were recorded for 415 individuals. The whole data-set was analyzed by Spearman correlation and multivariate statistical procedures, showing that albumin, serum alkaline phosphatase, haematocrit, and haemoglobin were the most significant functional markers of a flock's general conditions. Haematocrit and haemoglobin reflect animal health status, while albumin and serum alkaline phosphatase are a measure of nutritional status and physical activity, respectively. These are objective parameters, which can be easily measured from blood samples and have proved to be effective for grouping to interpret animal well-being.

Functional effect of haemoglobin polymorphism on the haematological pattern of Gentile di Puglia sheep.

The relationship between haemoglobin polymorphism and haematological pattern were studied in Gentile di Puglia, the Apulia Merino native breed. In a sample of 292 individuals, on five different farms, alpha- and beta-globin genetic polymorphism was investigated. A remarkable polymorphism was detected especially at alpha-globin gene where the variations concerned both the quality of the gene product and gene expression. Triplicated and quadruplicated alpha-globin genes were observed in 8.6% and 1.2% of the alpha-haplotypes respectively. At beta-globin (HBB) locus, predominance of HBB(B) allele was found in all flocks, while HBB(A) overall frequency was around 11.2%. Moreover, the effect of genotypes at globin systems on haematological data was evaluated on 289 animals. A significant effect was detected for HBB locus on haematocrit (HCT) and mean corpuscular volume (MCV), with decreasing HCT and MCV for decreasing number of HBB(A) alleles in the genotype. The opposite trend was observed for mean corpuscular haemoglobin concentration. The advances presented by this paper give reasons for inferring that BB genotypes can be expected to be more resilient to climatic stress and to local parasites.

In vitro inhibition of thymulin production in mercury-exposed thymus of young mice.

Lymphocyte differentiation, maturation and peripheral functions are affected by the thymic protein hormone thymulin. Mercury at very low concentrations has been seen to impair some lymphocytic functions causing subclinical manifestations in exposed workers. The present study was performed to test in vitro the effect of mercury on the production kinetics of thymulin using cultures of whole thymuses from young mice. Exposure to mercury (10(-8) M and 10(-6) M) added to the cultures, reduced kinetic thymulin production at all time intervals considered (1, 2, 4, 5 and 6 h) as compared to kinetic thymulin production of thymuses from young control mice. After the first hour the inhibition is more evident at the highest mercury concentration. Thymulin production decreased by 70, 74, 82 and 86% and by 55, 66, 73 and 81% for mercury concentrations of 10(-6) M and 10(-8) M, respectively, after 2, 4, 5 and 6 h. Mercury toxic effect on thymulin kinetics may be directly exerted to thymulin synthesis in epithelial cells, although it is less dramatic than that of cycloheximide (CHX), known as a potent inhibitor of protein synthesis in such cells. The toxic effect of mercury on thymic endocrine activity might cause the subclinical effects on cell-mediated immunological status observed in mercury exposed workers.

Identical marker alleles in Podolic cattle (Bos taurus) and Indian zebu (Bos indicus).

In the context of biochemical marker research and in order to add new information on native breeds, the present work focuses on a local Southern Italy cattle, namely Italian Podolic. We provide the complete structural characterisation of alpha-lactalbumins and beta-globin chains isolated from Podolic cattle (Bos taurus). Given the unavailability of the complete sequence for alpha-lactalbumin A of taurine cattle in the literature, we intended to check its structure in order to ascertain the absence of any possible silent mutation. Screening the Podolic cattle, we found a new beta-globin variant not detectable by conventional methods. The presence of such a new variant might be helpful in the study of the Podolic population genetic structure and for a better knowledge of the gene pool per se, and in comparison with the other breeds. Structural analyses showed that the new beta-globin Podolic variant exhibited the same sequence as beta-globin Azebu. The alpha-lactalbumin A was the same as that isolated from zebu cattle (Bos indicus). The results are discussed in relation to the possible involvement of the two markers in the debate on the origin of the Podolic breed.

Genetics and tolerance to tick-borne diseases in South Italy: experience in studying native Apulian and exotic sheep breed.

Tick borne diseases (TBDs), though neglected, are possibly the major constraints for livestock and agricultural production in southern Italy. The diseases affect mainly the livestock imported from foreign Northern countries. But now, very timidly indeed, the awareness that the use of native breeds could contribute to reduce the ravages of these diseases is growing. Over a period of many centuries Apulian farm animals have naturally developed the ability to thrive in TBDs endemic areas. This trait, which can be defined as tolerance to TBDs is associated with the ability to control parasitaemia and to resist the development of anaemia in the face of infection. It is a matter of fact that the hematological features of Apulian native farm animals are rather peculiar as the authors highlighted by studying the blood of these animals over a period of more than ten years. In the light of their experience the authors considered that the phenomenon of tolerance is a broad-based one and possibly not unrelated to the erythropoietic system of the Apulian animals. Thus the ability to resist anaemia per se was tested in four Altamurana sheep compared to that of four Romanov sheep. This work summarises the results obtained from the experimental anaemization of the above two breed groups and discusses the findings on the basis of a review of the authors' experience in studying TBDs in sheep.

Bovine hemoglobin alpha-globin chain polymorphism: primary structure determination of two new genetic variants by mass spectrometry and amino acid sequencing.

The present work describes the biochemical procedures used to identify the cause of a quantitative and qualitative hemoglobin polymorphism found in Podolian cattle. First, to analyze the different phenotypes, isoelectric focusing (IEF) of hemoglobins and RP-HPLC of globin chains was carried out; secondly, to determine accurately the globin molecular masses, electrospray mass spectrometry was performed and finally to check the entire amino acid sequences of the proteins, several enzymatic digests were analyzed by fast atom bombardment mass spectrometry (FAB-MS) and Edman degradation procedure. As to the qualitative polymorphism, the results of RP-HPLC show the presence of two alpha-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15,026.47 +/- 0.44 Da and 15,079.86 +/- 0.66 Da and whose respective primary structure differed from that of the common alpha-globin chain in the amino acid substitution Asn-->Ser at position 131 and the other in the replacement of the histidine residue at position 89 with tyrosine. As to the quantitative polymorphism, on the basis of the expression gradient found out in the duplicated alpha genes of several mammals, we conceive that the alpha 89 His-->Tyr is an allelic form of the I alpha gene while the alpha 131Asn-->Ser is an allelic form of the II alpha gene.

Expression gradient in sheep alpha alpha and alpha alpha alpha globin gene haplotypes: mRNA levels.

We previously found that in sheep alpha alpha-, alpha alpha alpha-, and alpha alpha alpha alpha-globin gene haplotypes, in which individual genes encode distinct allelic variants, the gene expression determined at protein level progressively decreases from the 5' to the 3' end. In the present study, through direct DNA analysis, we definitively established the gene relative position in the cluster and we verified the occurrence of the gradient also at the mRNA level. We measured the relative abundance of the alpha 113His-mRNA in alpha alpha and alpha alpha alpha haplotypes, in which the gene coding for the alpha 113His chain occupies the second and the third position, respectively. The alpha 113His-mRNA levels were about 20% and 8% to 9%, respectively, which closely paralleled alpha-chain levels. We conclude that the expression gradient occurs also at the mRNA level and is most likely of transcriptional origin.

Detection by reversed-phase HPLC of trace amounts of the alpha 113His globin chain in a sheep homozygous for a quadruple alpha-globin gene chromosome.

1. In sheep alpha alpha 113His and alpha D alpha alpha 113His globin gene haplotypes, the percentage gene efficiencies, from the 5' to the 3' end, are about 32-18 and 30-14-6, respectively. 2. We previously found that in a alpha alpha alpha alpha homozygote there may be a 1-2% alpha 113His chains, which however were difficult to resolve from alpha 113Leu chains by CMC chromatography. 3. We report here the experimental conditions which allowed a neat resolution of the 1% alpha 113His chain peak by RP-HPLC. 4. This finding strongly suggests the occurrence of the alpha D alpha alpha alpha 113His haplotype where the percentage gene efficiencies are 30-(14-5)-1, supporting the existence of a gradient of decreasing expression in multiple alpha-globin gene haplotypes.

Haemoglobin I: a new beta-globin chain variant found in sheep of Italian breeds.

A rather common haemoglobin variant was detected in the Sardinian and Altamurana sheep breeds. The mutated globin chain appears to be produced under the control of an allele at the HBB locus and due to a neutral amino acid substitution. The variant will be provisionally referred to as the Hb I.

DNA polymorphism at the casein loci in sheep.

By using seven endonucleases and four bovine cDNA probes specific for alpha S1-, alpha S2-, beta-, and kappa-casein genes, nine restriction fragment length polymorphisms (RFLPs) have been found in the sheep orthologous DNA regions. In contrast to the low level of variation observed at the protein level, these DNA polymorphisms determine a high level of heterozygosity and, therefore, represent useful tools for genetic analyses since they can also be obtained without the need for gene expression. In fact, informative matings suggest that in sheep, as in cattle, the four loci are linked.

Expression of triplicated and quadruplicated alpha globin genes in sheep.

In the sheep alpha alpha alpha globin gene haplotype, the three genes display from the 5' to the 3' end the percentage efficiencies of about 30:14:6, as indicated by the amounts of the three types of alpha chain produced in the alpha alpha alpha/alpha alpha alpha homozygotes. The 3' gene in the alpha alpha alpha alpha haplotype appears to have an efficiency around 1%, as suggested by analysis of one quadruple alpha homozygote. Moreover, the total outputs of the alpha alpha alpha as well as of the alpha alpha alpha alpha haplotypes do not substantially differ from that of the common alpha alpha haplotype.

Developmental G6PD polymorphism in Drosophila melanogaster: evidence for non-structural variants.

G6PD isozyme variation in Drosophila melanogaster is investigated in the larval stage through electrophoretic and genetic analyses. As current structural models for this gene-enzyme system fail to apply in these laboratory populations, the authors suggest a regulatory hypothesis to explain their observations.