Attention bias and novel object test in rams (Ovis aries) under intensive farming.

Affective states are long lasting mood states resulting from an accumulation of experiences. The knowledge of the affective state of animals can significantly help maintain and/or increase animal welfare. The aim of the study was to recognize the affective state of 13 adult rams reared under an intensive system and to further associate their affective state with hierarchy, sociability order and maintenance and social behaviour. The affective state was estimated by exposing them to an attention bias test and two novel object tests. Individuals with negative affective states performed reduced head hanging (p = 0.014), reduced agonistic behaviour (p = 0.033), increased social proximity (p = 0.009) and received less affiliative behaviours (p = 0.006). The study estimated the affective state of the rams and indicated easily recognizable maintenance and social behaviours correlated with negative affective state. Such behaviours could increase the awareness of the animals' welfare and thus improve management practices.

Genetic basis of ear length in sheep breeds sampled across the region from the Middle East to the Alps.

Ear length in sheep (Ovis aries) shows a wide range of natural variation, from the absence of an outer ear structure (anotia), to small outer ears (microtia), to regular ear length. Up until now, the underlying genetics of this phenotype has been studied in four sheep breeds from China, Jordan and Italy. These studies revealed a broad range of genes significantly associated with ear length, potentially indicating genetic heterogeneity across breeds or geographic regions. In the current study, we performed genome-wide SNP genotyping and haplotype-based mapping, in a population of 340 individuals, to identify loci influencing ear length variation in additional sheep breeds from Slovenia, Croatia, Cyprus and Greece. Additionally, two previously described candidate variants were also genotyped in our mapping population. The mapping model without candidate variant genotypes revealed only one genome-wide significant signal, which was located next to HMX1 on OAR6. This region was previously described as being associated with ear length variation in the Altay and Awassi sheep breeds. The mapping model including the candidate duplication genotype near HMX1 as a fixed effect explained the phenotypic variance on OAR6 and revealed an additional genome-wide significant locus on OAR13 associated with ear length. Our results, combined with published evidence, suggest that a duplication in the evolutionarily conserved region near HMX1 is the major regulator of ear length in sheep breeds descended from a larger region from Central Asia, to the Middle East, Cyprus, Greece and to the Alps. This distribution suggests an ancient origin of the derived allele.

Whole-genome SNP genotyping unveils ancestral and recent introgression in wild and domestic goats.

After the domestication of goats around 10,000 years before the present (BP), humans transported goats far beyond the range of their wild ancestor, the bezoar goat. This brought domestic goats into contact with many wild goat species such as ibex and markhor, enabling introgression between domestic and wild goats. To investigate this, while shedding light on the taxonomic status of wild and domestic goats, we analysed genome-wide SNP data of 613 specimens from 14 taxonomic units, including Capra hircus, C. pyrenaica, C. ibex (from Switzerland, Austria, Germany and Slovenia), C. aegagrus aegagrus, C. a. cretica, C. h. dorcas, C. caucasica caucasica, C. c. severtzovi, C. c. cylindricornis, C. falconeri, C. sibirica sibirica, C. s. alaiana and C. nubiana, as well as Oreamnos americanus (mountain goat) as an outgroup. To trace gene flow between domestic and wild goats, we integrated genotype data of local goat breeds from the Alps as well as from countries such as Spain, Greece, Türkiye, Egypt, Sudan, Iran, Russia (Caucasus and Altai) and Pakistan. Our phylogenetic analyses displayed a clear separation between bezoar-type and ibex-type clades with wild goats from the Greek islands of Crete and Youra clustered within domestic goats, confirming their feral origin. Our analyses also revealed gene flow between the lineages of Caucasian tur and domestic goats that most likely occurred before or during early domestication. Within the clade of domestic goats, analyses inferred gene flow between African and Iberian goats. The detected events of introgression were consistent with previous reports and offered interesting insights into the historical relationships among domestic and wild goats.

Ovine vocal individuality expression by ewes and lambs at a late (40 days) post-partum time point.

Recognizing the identity of conspecifics is important for survival and social interactions. In sheep, vocal individuality enhances postnatal recognition and strengthens the mother-offspring bond. Although previous studies report vocal individuality in an early postnatal period (3-15 days of life), scarce information exists on whether individuality occurs at a later postnatal time point. The aim of the study was to identify whether individuality in acoustic cues is expressed in ewes' and their lambs' bleats at 40 days post-partum. Dairy ewes (N = 21) and their suckling offspring (n = 30) were isolated separately without hearing or seeing each other, and vocalizations were recorded. Different approaches for estimating individuality on 18 determined acoustic parameters were implemented. All parameters showed individuality, but higher individuality appeared in those related with source and spectral characteristics. A discriminant function analysis showed similar levels of individuality between ewes and lambs, but lower than that reported in an earlier postnatal time, suggesting that ewes and lambs do not need strong individualized cues at the examined time point. In both cases, jitter was the only common parameter, suggesting its importance. Distinctive cues were noted between siblings. Ewes displayed vocal individuality at their dry season (a later time point than suckling period), which was based on amplitude-related acoustic parameters.

Large-scale mitogenome sequencing reveals consecutive expansions of domestic taurine cattle and supports sporadic aurochs introgression.

The contribution of domestic cattle in human societies is enormous, making cattle, along with other essential benefits, the economically most important domestic animal in the world today. To expand existing knowledge on cattle domestication and mitogenome diversity, we performed a comprehensive complete mitogenome analysis of the species (802 sequences, 114 breeds). A large sample was collected in South-east Europe, an important agricultural gateway to Europe during Neolithization and a region rich in cattle biodiversity. We found 1725 polymorphic sites (810 singletons, 853 parsimony-informative sites and 57 indels), 701 unique haplotypes, a haplotype diversity of 0.9995 and a nucleotide diversity of 0.0015. In addition to the dominant T3 and several rare haplogroups (Q, T5, T4, T2 and T1), we have identified maternal line in Austrian Murbodner cattle that possess surviving aurochs' mitochondria haplotype P1 that diverged prior to the Neolithization process. This is convincing evidence for rare female-mediated adaptive introgression of wild aurochs into domesticated cattle in Europe. We revalidated the existing haplogroup classification and provided Bayesian phylogenetic inference with a more precise estimated divergence time than previously available. Occasionally, classification based on partial mitogenomes was not reliable; for example, some individuals with haplogroups P and T5 were not recognized based on D-loop information. Bayesian skyline plot estimates (median) show that the earliest population growth began before domestication in cattle with haplogroup T2, followed by Q (~10.0-9.5 kyBP), whereas cattle with T3 (~7.5 kyBP) and T1 (~3.0-2.5 kyBP) expanded later. Overall, our results support the existence of interactions between aurochs and cattle during domestication and dispersal of cattle in the past, contribute to the conservation of maternal cattle diversity and enable functional analyses of the surviving aurochs P1 mitogenome.

Evaluation of Greek Cattle Carcass Characteristics (Carcass Weight and Age of Slaughter) Based on SEUROP Classification System.

In Greece, all cattle carcasses produced from a variety of breed types are classified according to the SEUROP system. The objective of this study was to evaluate Greek carcass characteristics such as carcass weight and age of slaughter based on SEUROP classification system (muscle conformation and fat deposit classes) and to describe the effect of main factors such as breed, gender, year of slaughter, farm's geographical region and month of slaughter on these carcass parameters. It is the first study that evaluates local breeds, revealing the wide diversity of the Greek cattle breeding conditions. The analyzed records consisted of 323,046 carcasses from 2011 to 2017. All the examined factors significantly affected the mean carcass weight (298.9 ± 0.2 kg) and the mean slaughter age (559.1 ± 0.3 days). Carcasses from beef meat breeds had on average higher mean carcass weight while the local breeds had lower. The mean slaughter age and carcass weight were higher in winter than in summer. The local and the dairy breeds were classified in similar muscle conformation classes. Finally, Greek cattle carcasses from almost all regions were satisfactory for their quality carcass traits with good muscle conformation (R, O and U class) and low-fat deposit (class 1 to 3).

Genomic diversity and population structure of the indigenous Greek and Cypriot cattle populations.

BACKGROUND: The indigenous cattle populations from Greece and Cyprus have decreased to small numbers and are currently at risk of extinction due to socio-economic reasons, geographic isolation and crossbreeding with commercial breeds. This study represents the first comprehensive genome-wide analysis of 10 indigenous cattle populations from continental Greece and the Greek islands, and one from Cyprus, and compares them with 104 international breeds using more than 46,000 single nucleotide polymorphisms (SNPs). RESULTS: We estimated several parameters of genetic diversity (e.g. heterozygosity and allelic diversity) that indicated a severe loss of genetic diversity for the island populations compared to the mainland populations, which is mainly due to the declining size of their population in recent years and subsequent inbreeding. This high inbreeding status also resulted in higher genetic differentiation within the Greek and Cyprus cattle group compared to the remaining geographical breed groups. Supervised and unsupervised cluster analyses revealed that the phylogenetic patterns in the indigenous Greek breeds were consistent with their geographical origin and historical information regarding crosses with breeds of Anatolian or Balkan origin. Cyprus cattle showed a relatively high indicine ancestry. Greek island populations are placed close to the root of the tree as defined by Gir and the outgroup Yak, whereas the mainland breeds share a common historical origin with Busa. Unsupervised clustering and D-statistics analyses provided strong support for Bos indicus introgression in almost all the investigated local cattle breeds along the route from Anatolia up to the southern foothills of the Alps, as well as in most cattle breeds along the Apennine peninsula to the southern foothills of the Alps. CONCLUSIONS: All investigated Cyprus and Greek breeds present complex mosaic genomes as a result of historical and recent admixture events between neighbor and well-separated breeds. While the contribution of some mainland breeds to the genetic diversity pool seems important, some island and fragmented mainland breeds suffer from a severe decline of population size and loss of alleles due to genetic drift. Conservation programs that are a compromise between what is feasible and what is desirable should focus not only on the still highly diverse mainland breeds but also promote and explore the conservation possibilities for island breeds.

The effect of extended milking intervals (24, 48 and 72 h) on milk yield, milk composition, mammary physiology and welfare traits in dairy ewes.

The majority of the work today regarding the effects of extended milking intervals has focused on dairy cattle and only to a limited extent on dairy goats and sheep. The aim of this study was to investigate the effect of different non-milking intervals on milk yield and composition, mammary physiology and welfare indices in dairy ewes. Thirty-six multiparous ewes in late lactation were allocated to one of four groups of nine and subjected to 24, 48 or 72 h of non-milking or normal milking interval (12 h) (group A, B, C and D, respectively). Data showed that there were no significant differences in milk yield among the experimental groups during the third day after re-milking. Furthermore, no significant differences in milk lactose, protein and fat concentration among the experimental groups were observed after 7, 14 and 21 d of re-milking, respectively. Non-milking for 72 h resulted in a temporary increase of sodium concentration, Na + /K + ratio and enzymatic activities of plasmin (PL), plasminogen (PG) and plasminogen activator (PA) in milk. However, these parameters had similar values among the experimental groups on day 5 after re-milking. The concentration of lactose in blood was also significantly increased as a result of the 72 h non-milking interval and returned to its initial levels the second day after re-milking. These data, taken together, suggest that early involution events that occurred as an effect of non-milking were fully reversible within a short period of time. Finally, no significant signs of welfare impairment were observed in ewes due to extended milking intervals. In conclusion, non-milking up to 72 h had no negative effects on milk yield and composition, mammary physiology and welfare parameters in dairy ewes.

Effects of gestational maternal undernutrition on growth, carcass composition and meat quality of rabbit offspring.

An experiment was conducted in order to evaluate the effects of gestational undernutrition of rabbit does on growth, carcass composition and meat quality of the offsprings. Thirty primiparous non lactating rabbit does were artificially inseminated and randomly divided in three treatment groups: Control (C; fed to 100% of maintenance requirements throughout gestation, n = 10), early undernourished (EU; fed to 50% of maintenance requirements during days 7-19 of gestation, n = 10) and late undernourished (LU; fed to 50% of maintenance requirements during days 20-27 of gestation, n = 10). During the 4th week of the gestation period, LU does significantly lost weight compared to C and EU groups (P<0.05). At kindling, C does produced litters with higher proportions of stillborn kits (P<0.05) while the total litter size (alive and stillborn kits) was not different among groups (10.7, 12.8 and 12.7 kits in C, EU and LU groups, respectively). Kit birth weight tended to be lower in the LU group. During fattening, body weight and feed intake were not different among offsprings of the three experimental groups. Moreover, the maternal undernutrition did not have any impact on carcass composition of the offsprings in terms of carcass parts and internal organs weights as well as meat quality of L. lumborum muscle (pH24, colour, water holding capacity and shear values) at slaughter (70 days of age). Therefore, it can be concluded that the gestational undernutrition of the mother does not have detrimental effects on the productive and quality traits of the offsprings.

Severe maternal undernutrition and post-weaning behavior of rabbits.

The objective of the present experiment was to investigate the implications of severe maternal undernutrition on the post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group (C) that was fed 100% of the recommended energy maintenance requirements throughout pregnancy and lactation and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirements between the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and between the 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages was recorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear and emotional distress. Significant differences were found between the U2 and the other experimental groups for the duration of eating and drinking, and duration of locomotory and investigatory behaviors (P<0.05). At the age of 65 days, duration of comfort behaviors was also lower in the U2 compared to the other groups (P<0.05). On the other hand, duration of resting appeared to be the highest in the group of rabbits born from undernourished does between the 20th and the 27th day of pregnancy (P<0.05). Duration of resting was significantly increased, although frequency and duration of eating and drinking were significantly decreased at the age of 65 days (P<0.05). Male rabbits had greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared to the females (P<0.01). A decreased activity of rabbits during the light period was observed (as expected), whereas an increase in duration of locomotory and investigatory behaviors was observed during the first four and the last 4h of the light and dark periods, respectively (P<0.05). Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start position compared to the other groups during the implementation of the open-field test (P<0.05). No other significant differences in the exhibition of the examined behaviors were found between the treatment groups, with the exception of 'moving forelegs' activity that had the lowest value in the U3 group of rabbits. It is concluded that severe maternal undernutrition (50% of the recommended maintenance requirements) especially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active compared to the other experimental groups.

Energy balance-dependent regulation of ovine glucose 6-phosphate dehydrogenase protein isoform expression.

G6PDH is the rate-limiting enzyme of the pentose phosphate pathway and one of the principal source of NADPH, a major cellular reductant. Importantly, in ruminant's metabolism the aforementioned NADPH provided, is utilized for de novo fatty acid synthesis. Previous work of cloning the ovine (Ovis aries) og6pdh gene has revealed the presence of two cDNA transcripts (og6pda and og6pdb), og6pdb being a product of alternative splicing not similar to any other previously reported.(1) In the current study the effect of energy balance in the ovine G6PDH protein expression was investigated, shedding light on the biochemical features and potential physiological role of the oG6PDB isoform. Changes in energy balance leads to protein expression changes in both transcripts, to the opposite direction and not in a proportional way. Negative energy balance was not in favor of the presence of any particular isoform, while both protein expression levels were not significantly different (P > 0.05). In contrast, at the transition point from negative to positive and on the positive energy balance, there is a significant increase of oG6PDA compared with oG6PDB protein expression (P < 0.001). Both oG6PDH protein isoforms changed significantly toward the positive energy balance. oG6PDA is escalating, while oG6PDB is falling, under the same stimulus (positive energy balance alteration). This change is also positively associated with increasing levels in enzyme activity, 4 weeks post-weaning in ewes' adipose tissue. Furthermore, regression analysis clearly demonstrated the linear correlation of both proteins in response to the WPW, while energy balance, enzyme activity, and oG6PDA relative protein expression follow the same escalating trend; in contrast, oG6PDB relative protein expression falls in time, similar to both transcripts accumulation pattern, as reported previously.(2.)

Increase of energy balance significantly alters major lipogenic gene expression in lactation ewes.

The objective of the present study was to examine changes observed in the expression of cytosolic NADP isocitrate dehydrogenase (ICDH) and glucose 6-phosphate dehydrogenase (G6PD) genes, the major implicated genes in ruminant lipogenesis in terms of produce NADPH, during the early post-weaning period in dairy ewes in respect to energy intake, and to further correlate the noted changes with their respective enzymatic activities. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating (2nd lactation period) dairy ewes of the Chios breed. Adipose tissue samples were taken from the tail head region at weeks 1, 2, and 4 after weaning (45 days after parturition). Dairy ewes were in negative energy balance during weeks 1 and 2 after weaning and they moved into a strong positive energy balance at week 4 after weaning. Expression of ICDH and G6PD genes and their respective enzymatic activity was determined. Results showed that both genes' expression and enzymatic activities were significantly minimal at week 1 after weaning, reaching a maximum level at week 4 after weaning (P < 0.05). A 3.5-fold and a 5-fold increase of G6PD and ICDH mRNA levels were observed, respectively. Concerning their respective enzymatic activities, a 5.5-fold and 2-fold increase was noted, respectively. A positive correlation was found between ICDH and G6PD gene expression (P < 0.001) indicating a synchronized response to energy intake changes. Almost similar changes were observed for enzymatic activities, rendering these enzymes as potential biochemical markers of ovine lipogenesis.

Enzymatic and mRNA Transcript Response of Ovine 6-Phosphogluconate Dehydrogenase (6PGD) in Respect to Different Milk Yield.

Ovine 6-phosphogluconate dehydrogenase (6PGD) is an enzyme of the pentose phosphate pathway, providing the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted both on enzymatic level and on molecular level. However, to our knowledge, any correlation between enzymatic activity and 6PGD gene expression pattern related to different physiological stages has not been yet reported. With this report, we tried to highlight if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to different milk yield. According to the determined enzymatic activities and adipocytes characteristics, ewes with low milk production possessed a greater (P ≤ .001) 6PGD activity and larger adipocytes than the highly productive ewes. Although 6PGD expression pattern was higher in low milk yield ewes than in ewes with high milk production, this difference was not found statistically significant. Thus, 6PGD gene expression pattern was not followed by so rapid and great/sizeable changes as it was observed for its respective enzymatic activity, suggesting that other mechanisms such as post translation regulation may be involved in the regulation of the respective gene.

Enzymatic and mRNA transcript response of ovine 6-phosphogluconate dehydrogenase (6PGD) in respect to different weights from weaning to four months of age.

Ovine 6-phosphogluconate dehydrogenase (6PGD), an enzyme of the pentose phosphate pathway, provides the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted not only on the enzymatic level, but also on molecular level elucidating its cDNA sequence. Herein, we tried to elucidate if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to two different weights from weaning to 4 months old. 18 male and 16 female lambs of Chios breed were randomly selected after weaning and assigned to two groups based on sex in a different experimental open-plan shed. Two subgroups were defined in each sex and they were slaughtered at 25 kg and 30 kg, respectively. Samples of adipose tissue (tail, perirenal and shoulder site) were collected and 6PGD enzymatic activity, gene expression, and characteristics of adipocytes were determined. According to the determined data, tail subcutaneous adipose tissue matures later than the others examined tissues and has a diminished lipogenic activity. A 6PGD gene expression pattern was not followed by analogous changes of its enzymatic activity, suggesting that other mechanisms such as post transcription or/and post translation regulation may be involved.

Molecular study of ovine glucose 6-phosphate dehydrogenase gene expression in respect to different energy intake.

Glucose 6-phosphate dehydrogenase (G6PD) plays an important role in a ruminant's metabolism catalyzing the first committed reaction in the pentose phosphate pathway as it provides necessary compounds of NADPH for the synthesis of fatty acids. The cloning of ovine (Ovis aries) G6PD gene revealed the presence of two cDNA transcripts (oG6PD(A) and oG6PD(B)), with oG6PD(B) being a product of alternative splicing and with no similarity to any other previously reported G6PD transcript. Here, we attempt to study the effect of energy balance in ovine G6PD transcript expression, trying simultaneously to find out any potential physiological role of the oG6PD(B) transcript. Changes of energy balance that lead to synergistic changes in the expression of both transcripts, but in opposite directions and not in a proportional way. Negative energy balance favours the presence of the oG6PD(B) transcript leading to a significant increase of its expression, compared to oG6PD(A) expression (P<0.05). In contrast, positive energy balance leads to a significant increase of oG6PD(A) compared to oG6PD(B) expression (P<0.05). In either condition oG6PD(B) expression is unchanged. Regression analysis showed that there is an energy balance threshold where the expression of both transcripts shows no change.

The ovine plasminogen activator inhibitors type 1 and type 2 cDNAs: molecular cloning, characterization and expression in various tissues.

Two serine proteases, urokinase and tissue type, control the activation of plasminogen to plasmin. These proteases are in turn specifically inhibited by plasminogen activator inhibitors type 1 and 2 (PAI-1 and PAI-2), both of which belong to the serine protease inhibitor (serpin) superfamily. Very little information is available on the role of PAI-1 and PAI-2 in ruminants, in mammary gland involution and in the adipose tissue. In this paper we describe the isolation of the full-length cDNAs of ovine PAI-1 and PAI-2 using a polymerase chain reaction based strategy. The ovine PAI-1 cDNA comprised of 1460bp and it is characterized by a coding region of 1209bp, and 5'- and 3'-UTR regions of 147 and 104bp, respectively. The deduced amino acid sequence consists of 402 amino acids. The ovine PAI-2 cDNA is comprised of 2128bp and it is characterized by a coding region of 957bp and 5'- and 3'-UTR regions of 58 and 819bp respectively. The deduced amino acid sequence consists of 416 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed that the two inhibitors were predominantly expressed in the ovine mammary gland and adipose tissue. Furthermore, PAI-1 and PAI-2 mRNA levels were higher in the involuting mammary tissue and the adipose tissue obtained from non-lactating ewes compared to the corresponding values in tissues obtained from lactating ewes. These data are consistent with the notion that the plasminogen activation cascade plays a key role in involution of the mammary gland. The upregulation of expression of both inhibitors in the adipose tissue during the non-lactating period is a rather enigmatic observation. However, the expression of both inhibitors (PAI-1 and PAI-2) together with that of urokinase type plasminogen activator and its receptor previously reported by our group, strengthen the suggestion that the adipose tissue functions as an endocrine besides an energy storage organ.

The ovine urokinase plasminogen activator and its receptor cDNAs: molecular cloning, characterization and expression in various tissues.

The activation of plasminogen plays a crucial role in a variety of extracellular proteolytic events such as, fibrinolysis, cell migration, ovulation, involution of the mammary gland and the activation of other protease classes and growth factors. In this paper we describe the isolation of the full-length cDNAs of ovine urokinase plasminogen activator (u-PA) and its receptor (u-PAR) using a polymerase chain reaction based strategy. The ovine u-PA cDNA comprised of 2350 bp and it is characterized by a coding region of 1302 bp, and 5'- and 3'-UTR regions of 129 and 919 bp, respectively. The deduced amino acid sequence consists of 433 amino acids. The ovine u-PAR cDNA is comprised of 1247 bp and it is characterized by a coding region of 957 bp and 5'- and 3'-UTR regions of 44 and 246 bp respectively. The deduced amino acid sequence consists of 318 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed high levels of u-PA expression in the adipose tissue, followed by that in mammary gland and kidney. Lower levels of expression were detected in the adrenal glands, heart, ovaries, spleen, liver and cerebellum. A similar pattern was observed in u-PAR expression with noticeably lower levels of expression in heart, liver and cerebellum. To the best of our knowledge, this is the first paper reporting expression of u-PA and u-PAR in the adipose tissue. These data strengthen the suggestion that adipose tissue functions as an endocrine organ besides an energy storage organ. Furthermore, u-PA and u-PAR mRNA levels were 7 and 8.5 fold higher respectively in involuting mammary tissue obtained from non-lactating ewes compared to that detected in mammary tissue obtained from lactating ewes. These data are consistent with the notion that upregulation of u-PA and u-PAR expression may play a key role in the process of involution of the mammary gland.

Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the -140/+18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5' flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines.

Molecular cloning and characterization of the sheep malic enzyme cDNA.

Malic enzyme catalyzes decarboxylation of malate to pyruvate and CO(2), providing de novo biosynthesis of fatty acids with NADPH. Since lipogenesis in ruminants, contrarily to some monogastric species like human and rodents, occurs predominantly in adipose tissue, the activity of many lipogenic enzymes is higher in adipose tissue compared to liver. Expression of malic enzyme is regulated by nutrition; refeeding after a period of starvation results to an induction of the enzyme. Here we present the nucleotide sequence of two transcripts of the ovine cytosolic malic enzyme gene that differ at the length of the 3' UTR. These are the first published cDNA sequences for ruminant species and share high similarity with the corresponding sequences of other species. Malic enzyme mRNA was present in every ovine tissue that was examined. In agreement with the fact that adipose tissue is the major lipogenic site for ruminants, mRNA levels in adipose tissue were higher than in liver. Refeeding after two weeks of caloric restriction resulted in a two-fold increase of the mRNA level of malic enzyme in adipose tissue.

Cloning and functional characterization of the ovine Hormone Sensitive Lipase (HSL) full-length cDNAs: an integrated approach.

Hormone Sensitive Lipase (HSL) is a highly regulated enzyme that mediates lipolysis in adipocytes. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signalling cascade reactions. Since HSL constitutes the key enzyme in the regulation of lipid stores and the only enzyme being subjected to hormonal regulation [in terms of the recently identified Adipose Triglyceride Lipase (ATGL)], the ovine Hormone Sensitive Lipase (ovHSL) full-length cDNA clones were isolated, using a Polymerase Chain Reaction-based (PCR) strategy. The two isolated isoforms ovHSL-A and ovHSL-B contain two highly homologous Open Reading Frame (ORF) regions of 2.089 Kb and 2.086 Kb, respectively, the latter having been missed the 688th triplet coding for glutamine (DeltaQ(688)). The putative 695 and 694 amino acid respective sequences bear strong homologies with other HSL protein family members. Southern blotting analysis revealed that HSL is represented as a single copy gene in the ovine genome, while Reverse Transcription-PCR (RT-PCR) approaches unambiguously dictated its variable transcriptional expression profile in the different tissues examined. Interestingly, as undoubtedly corroborated by both RT-PCR and Western blotting analysis, ovHSL gene expression is notably enhanced in the adipose tissue during the fasting period, when lipolysis is highly increased in ruminant species. Based on the crystal structure of an Archaeoglobus fulgidus enzyme, a three-dimensional (3D) molecular model of the ovHSL putative catalytic domain was constructed, thus providing an inchoative insight into understanding the enzymatic activity and functional regulation mechanisms of the ruminant HSL gene product(s).