Seasonality in sheep reproduction and related limitations make milk production challenging throughout the year. In the present study, we investigated the response to estrus induction treatments in three indigenous breeds, Florina, Chios, and Karagouniko, as well as the melatonin receptor 1A gene variants in relation to this response. The three distinct synchronization methods were A: intravaginal sponges, B: GNRH use, and C: male effect. In group A, fertility was 85%, and Florina ewes expressed estrus at 90% in July. Ewes from Karagouniko and Chios had fecundity rates of 95% and 99%, respectively, and 100% estrus expression. The Florina ewes in group B expressed estrus at a percentage of 60%, with a fecundity rate of 57%, the Karagouniko ewes at a percentage of 65%, with a fecundity rate of 54%, and the Chios breed animals at a percentage of 87%, with a fecundity rate of 85%. Twenty to twenty-five days after ram induction, 68% of the Florina breed in group C showed signs of estrus, compared to 84% and 94% of Karagouniko and Chios breeds, respectively. In both Florina and Karagouniko breeds, all treatments showed a substantial difference in the frequency of the four identified SNPs in the MTNR1A gene between ewes who expressed estrus and ewes who did not. The genetic improvement based on the alleles analyzed in the current study is expected to decrease seasonality rates in indigenous sheep breeds.

The aim of this study was to evaluate the effects of two different methods of controlling ovine reproduction (ram effect and a combination of eco-friendly substances) in three Greek indigenous sheep breeds, namely Florina, Karagouniko, and Chios. 180 ewes, aged 2–3 years old, were used, equally divided between the three Greek sheep breeds. Each breed was divided in two equal experimental groups: In group ECO (combination of eco-friendly substances), a dose of Gonadotropin-releasing hormone (GnRH) was administered at day 0, followed by an intramuscular injection of prostaglandin seven days later and another dose of GnRH at day 9. In group ME, sexually active males were introduced to the females that have been isolated for 3 months. The experiment was conducted in three consecutive years: 2020, 2021, and 2022. Average estrus expression and fecundity in all three examined breeds was comparable, with the exemption of Florina ewes treated with the eco-friendly substances in the first year, a result that could be attributed to the strong seasonal pattern in estrus expression of the Florina breed. The average length to estrus expression in days was significantly lower in the eco-friendly method than the procedure using male effect and the prolificacy was higher for the Chios sheep than the Florina and the Karagouniko breeds. Overall, both methods for the induction and synchronization of estrus (male effect and PGF2a administration) could be used in sheep reproduction with promising results. Nevertheless, the male effect seems to be more efficient in indigenous breeds with long anestrus periods.

Small ruminants are characterised by large anoestrus periods in temperate latitudes due to photoperiodism. This is a major obstacle in sheep and goat farming in several countries where milk from these animals constitute important products for national economies. In an effort to reduce the usage of exogenous hormones administration for oestrus induction and there reproduction and milk production throughout the year, genetic improvement scoping to non seasonal dependent breeding could be a promising alternative. Keeping this in mind, this mini review presents the most commonly studied genes that have been associated with reproduction seasonality in small ruminants, as well as and most importantly the successful selective breeding schemes that have achieved reproduction seasonality elimination. Although numerous genes and Single Nucleotide Polymorphisms (SNPs) have been associated with seasonality as listed here, they have been scarcely utilised in selective breeding programs, calling their efficiency as selective breeding targets into question. Future research should, hence, evaluate chronic data from implementation of selective breeding schemes in order to facilitate their validity in various breeds.

Sheep farming plays a crucial role for Mediterranean countries, wherefrom a plethora of dairy products is produced. Yet, numerous indigenous sheep breeds in temperate latitudes such as the Mediterranean present a serious drawback that milk production is impaired by the seasonality of reproduction. Towards the efforts for reduction of this phenomenon, the purpose of this study was to evaluate and compare different oestrus synchronization treatments, as well as to associate two genes alleles', namely the MTNR1A and the GDF9, with the response to those treatments. Three indigenous breeds were investigated (Florina, Chios and Karagouniko sheep) and inside each breed three different oestrus synchronization treatments were applied (A: intravaginal sponges, B: GNRH use and C: male effect). In group A, Florina ewes expressed oestrus at 90% in July and fecundity was 85%. Karagouniko and Chios ewes exhibited an oestrus expression of 100% with fecundity rates at 95% and 99%, respectively. In group B, Florina ewes expressed oestrus at 60% with fecundity at 57%, Karagouniko ewes expressed oestrus at 65% with fecundity at 54%, whereas Chios breed animals expressed oestrus at 87% with fecundity rate at 85%. In group C, 68% of the Florina breed expressed oestrus 20-25 days post ram induction, whereas this proportion was 84% and 94%, for Karagouniko and Chios breed, respectively. For the molecular analysis, partial segments of the two genes were sequenced and analysed, whereas alleles were scored based on the detected SNPs. All frequencies of the four detected SNPs in MTNR1A gene were statistically and significantly different in ewes that expressed oestrus in comparison with ewes that did not express oestrus in Florina and Karagouniko breeds concerning all treatments. Two SNP's were detected in GDF9 gene, G1 and Fec(G), from which, only the Fec(G) mutation exhibited statistically significant difference in twins and triplets than in singles in Florina and Karagouniko breeds.

Greece has a long history in autochthonous sheep, the genetic ancestry of which has been associated with four subtypes known to inhabit Greece at the end of the nineteenth century. Among them, the Karamaniko breed is still surviving, however endangered. This study was designed in order to (a) determine the phylogenetic status, (b) to evaluate the levels of inbreeding, and (c) to assess the genetic basis of coat color of Karamaniko breed. For these purposes, the mitochondrial cyt b gene was sequenced, the AFLP methodology was applied, and the MC1R was genotyped, respectively, in 72 female sheep from the Karamaniko breed. Four different novel cyt b haplotypes were defined and three MC1R genotypes were scored, whereas inbreeding levels estimated using AFLPs by the means of relatedness coefficient (r) were 0.287, with gene diversity at the levels of 0.105. Phylogenetic analysis indicated an eastern Asian tropical and subtropical origin of the Karamaniko breed, close with breeds originating from central Turkey, or a clustering within western European or Mediterranean sheep, mirroring a recent genetic divergence, with a non-random spread towards the formation of lowland breeds. The MC1R genotypes were all associated with the white coat color, in which selective breeding has probably been based on traditional morphological characters. Finally, levels of inbreeding do not constitute an indication for a particular mating plan to prevent unpleasant phenomena such as inbreeding depression, probably because of the special attention paid by the farmers towards the avoidance of relative recurrent mating.

Early and reliable diagnosis of pregnancy in sows is considered one of the most crucial parameters for the proper management in pig farming. Reproductive activity of the sows is directly correlated with the overall productivity of the enterprise. The best and most precise methodology to diagnose pregnancy in farm animals, including pigs, is ultrasonography. For getting optimal outputs of real-time ultrasonography application in pigs, both gestation status and litter size of sows is of high importance. Hence, a detailed evaluation and careful reading of the retrieved ultrasonic image could serve to the prediction of the sow’s litter size, providing valuable information to the producer. In the present article we studied the ultrasonic typification of sows in an effort to develop a methodology for the precise pregnancy diagnosis and the prediction of the litter size. To achieve this goal, 1214 matings and 1010 pregnancies were recorded and evaluated over a period of 20 months, in a pig unit in Northern Greece. Two thorough scans were performed on each sow, classifying the ultrasonic image in a scale of 0 to 10, 0 being ‘non pregnant’ and 10 given to the most reassuring image of pregnancy. Evaluations took place in a wide range of time since mating, hence between days 16 (the earliest first scan) and 68 (the latest that a second scan was performed) and the mean values obtained were 8,36 and 8,83 for the first and second scan respectively. Furthermore, the litter size of each subsequent parturition was recorded (mean: 12.22) and correlated to the score of each of the two scans performed during pregnancy. The results showed that more reassuring images and higher scores for both examinations of pregnancy were achieved with increasing time interval from mating (P<0.05). It is therefore suggested that a detailed real-time ultrasonic scanning, can provide a very useful tool, not only for pregnancy diagnosis, but also for the prediction of litter size and eventually the precise management of pregnant sows.

Tendinopathies constitute a very common category of musculoskeletal disorders, causing economic losses in the equine industry and animal welfare issues in horse populations. Sport and racehorses are in general sensitive to tendinopathies, whereas local indigenous horse populations are often more tolerant to various disorders. Particularly, indigenous Greek horse breeds have evolved and adapted in the rough topographic features of mountainous and semimountainous Greek terrain and are less prone to develop tendinopathies. Susceptibility to tendinopathy has been proposed to be associated with three specific variants in the tenascin-C and collagen type 5 α-1 genes. The present study was designed to analyze these genes and estimate the frequency of the aforementioned variants in three indigenous Greek horse populations, namely the pinia (peneia) breed, the Messara breed, and the nondescript Macedonian pacer, in comparison with a population of warmbloods. Results revealed high frequencies of genotypes with high risk for tendinopathy in the warmblood population and high frequency of low risk genotypes in the Greek breeds. Apart from the three variants, two novel polymorphisms were detected, one of which may also be associated with risk for tendinopathy. Our findings (a) are in agreement with previous ones demonstrating that risk for tendinopathies is associated with particular gene variants and also (b) highlight the value of Greek indigenous horse breeds as a genetic resource that (c) may be used in selective breeding schemes for decreasing the risk for tendinopathies in genetically improved breeds.

Characterization of indigenous domestic animal breeds is of high importance in the sense of conservation and genetic improvement. There are six registered indigenous Greek horse breeds, defined by geographic orientation, as well as a few non-descript ones. Among the latter, the Macedonian pacer population is the most featured one, exhibiting the specific gaiting ability to pace. This study constitutes the first genetic investigation of the Macedonian pacer horses, in an effort to examine the validity of their inclusion in the herdbook as a distinct breed. To achieve this goal, we analyzed the DMRT3 gene, which has been associated with the ability to pace, as well as the mitochondrial cyt b gene, to estimate the genetic relationships with other breeds. Analysis of the DMRT3 gene revealed a very high frequency (0.988) of the “gait keeper” allelic mutation. Genetic differentiation of the Macedonian pacer horses based on cyt b sequencing was in almost all pairwise comparisons significant and in equal or greater levels than in pairs of the rest Greek breeds, with Fst ranging from 0 to 0.355. Haplotype and nucleotide diversity in the Macedonian pacer population were 0.917 and 0.0044, respectively. Phylogenetic analysis resulted in low genetic distances supported by not significant values that indicated slight genetic divergence among different breeds. Overall, the fact that Macedonian horses exhibit a particular phenotypic character of locomotion, transmitted to the next generations in combination with the significant levels of genetic differentiation, are considered valuable data toward their recognition and official registration.

The aim of the present study was to determine any potential association of the BF, RBP4, and ESR2 genes with reproduction traits in an autochthonous Greek pig population. The PCR-RFLP methodology was implemented for genotyping purposes of the examined genes. No deviation from the Hardy-Weinberg equilibrium was observed for the examined loci, while the B allele noted to be the more frequent in all analyzed genes. In addition, sows with the AA genotype of BF gene found to produce significantly lower numbers of the total born piglets (TNB) and number of piglets born alive (TNA), while the respective BB genotype significantly exceeded in TNB and NBA traits compared to the other two genotypes (P < 0.001). In the case of RBP4 gene, sows with the AB genotype noted to have significant higher values of TNB and NBA in regard to the respective homozygous genotypes (AA or BB), while the BB genotype showed intermediate results (P < 0.001). There were no statistical differences between the BB genotype and the AB genotype of ESR2 locus in regard to the examined traits. However, a noticeable superiority (P < 0.01) of the BB genotype compared to the homozygous AA genotype, adding almost 2 piglets/litter in TNB and NBA trait, was found. ABBREVIATIONS:TNB: Total number of born piglets; NBA: Number of piglets born alive.

PCR-RFLP was applied to a commercial crossbred pig population in order to investigate the association between polymorphism (SNP) of Retinol-binding protein 4 (RBP4) gene and reproductive performance. 400 sows were genotyped and 2000 records of reproductive traits were used in order to retrieve information about the allele frequencies and the association of the RBP4 gene with main reproductive characteristics of the population. A deviation from the Hardy-Weinberg equilibrium was observed as a result of the AB genotype excess. In addition, the AA genotype saw statistically significant higher values of (i) the total number of born piglets (p<0.05), (ii) the number of piglets born alive (p<0.01), and (iii) the number of weaned piglets (p<0.01). The number of the mummified piglets and the number of the piglets born dead did not differ between the various RBP4 genotypes. Interestingly, the AA genotype had a negative impact (p<0.05) on the number of piglets born dead, resulting indirectly in a larger litter size. In conclusion, the AA genotype and in extension the A allele of RBP4 gene are in favor of producing larger litter size, suggesting that the RBP4 gene may be used in Marker-Assisted Selection (MAS) programs for a rapid improvement of the reproductive characteristics in pigs.

Sheep’s reproductive physiology in temperate latitudes (such as Greece), is characterized by seasonality and is also regulated by photoperiodic exposure. Melatonin is the key hormone involved in this regulation. However, the melatonin secretion and therefore the ewes reproductive activity underlies variation, proposed to be linked with the melatonin receptor subtype 1A (MNTR1A) gene structure. This study was designed to investigate the polymorphism of the MNTR1A gene in a local Greek sheep breed and to determine its potential association with reproductive seasonality.

Toll-like receptors (TLRs) and b-defensins (BD) molecules are group of molecules that recognize various microbial components and play a crucial role in the activation of the innate immune system in vertebrate species. Although TLRs gene expression has been studied in various pig tissues, little is known about their expression in porcine reproductive tract. Concerning b-defensins genes, only BD1, 2 and 3 counterparts have been well studied in pigs' reproductive organs. The aim of this study was to investigate the expression pattern of both gene families in pigs' male and female reproductive organs, and embryos, as potential tool for further association studies in respect to immunity and disease resistance. RT-PCR analysis revealed that all of the examined TLR genes were expressed in the reproductive organs of male and female pigs, with TLR3 and TLR5 showing the higher levels and TLR9 the lowest, in all analyzed tissues. BD genes showed a different expression pattern in respect to the examined tissue. In embryos, TLR1 revealed high expression levels, while only BD3, BD108, and BD123 were found to be expressed.

Le projet a developpe des solutions innovantes pour fournir du lait de la chevre sans hormones toute au long de l'annee. FLOCK-REPROD, soutenu par 7eme Programme Cadre, proposees les solutions qui ne necessitent pas le traitement hormonal et permettent le controle de la saisonnalite et de l'application de l'insemination artificielle (IA). De cette maniere, FLOCK-REPROD aidera les eleveurs produisent plus de lait et de fournir un moyen alternatif de repondre aux exigences legales de l'UE qui restreint l'utilisation d'hormones. Les chercheurs ont elabores de nouveaux protocoles d’AI sans utilisation de gestagens (PG1, PG2, HF) impliquant des procedures basees sur l'effet mâle et traitements lumineuses pour induire et synchroniser des ovulations en toute saison d’annee. Protocoles FLOCK-REPROD excluent l'utilisation de gestagens. PG1 et PG2 sont bases sur 1 ou 2 injections de prostaglandine (pas encore soumis a des contraintes de residus). Le protocole HF est sans hormone, et peut donc etre appliquee dans des fermes organiques. De nouveaux protocoles d'IA ont ete testes dans des conditions de terrain. Les meilleurs resultats ont ete obtenus avec du protocole HF (58% de gestations, similaire au protocole hormonal classique HT), suivie par PG2 (54%) et PG1 (45%). Le probleme principal pour la mise en œuvre de protocoles de PG1 et HF etait grande variabilite des resultats de fertilite en fonction de fermes. De nouveaux protocoles d'IA etaient moins efficaces en termes de temps de travail et des couts des donnees, par rapport a la HT. PG1 est le protocole le plus prenant de temps, suivie d'HF et PG2. HF semblait etre le protocole le plus cher alors que PG2 etait moins cher que PG1. Charge de travail accrue et des couts plus eleves generes par de nouveaux protocoles d'IA etaient principalement en raison du nombre de boucs supplementaires necessaires pour mener l’effet mâle (couts plus eleves des alimentations, prenant du temps pour la manipulation avec les boucs).

The epididymis is an organ involved in the maturation, transport, and storage of sperm prior to ejaculation. As epididymis is exposed to a constant risk of inflammatory conditions that may lead to transient or permanent sterility, protection of this organ from pathogens is an essential aspect of reproductive physiology. The families of antimicrobial peptides β-defensins and the pattern-recognition receptors Toll-like (TLR) mediate innate immunity in various vertebrates including avian species. As rooster infertility is a major concern in the poultry industry, the objectives of this study were to determine the expression profile of the entire family of the avian β-defensins (AvBD) and TLR genes in the rooster epididymis, to investigate whether sexual maturation affects their epididymidal mRNA abundance and to determine the changes in their expression levels in response to Salmonella enteritidis (SE) infection in the epididymis of sexually mature roosters. RNA was extracted from the epididymis of healthy pubertal, sexually mature and aged birds, and from sexually mature SE infected birds. RT-PCR analysis revealed that 10 members of the AvBD and nine members of the TLR gene families were expressed in the epididymis. Quantitative real-time PCR analysis revealed that the epididymidal mRNA abundance of certain AvBD and TLR genes was developmentally regulated with respect to sexual maturation. SE infection resulted in a significant induction of AvBD 1, 9, 10, 12 and 14, as well as TLR 1-2, 2-1, 2-2, 4, 5 and 7 genes, in the epididymis of sexually mature roosters, compared to healthy birds of the same age. These findings provide strong evidence to suggest that the rooster epididymis is capable of initiating an inflammatory response to Salmonella, through activation of certain members of the AvBD and TLR gene families.

In the present study, the single nucleotide polymorphism of CFB (Complement Factor B), BF gene, was analyzed by PCR-RFLP in a commercial pig population in Greece. BF gene is coding for properdin, a protein that plays an integral role in the uterine epithelium growth and is found in a QTL region with many other genes associated with specific reproductive traits. BF gene is considered to affect various reproductive traits in pigs and the present study associated the BF gene genotypes with litter size of the sows (TNB, total number born and NBA number born alive piglets/birth). Sows with AB genotype gave statistically significant lower values for TNB and NBA piglets/birth (p<0.05) than the BB genotype. Our results support the concept that BF belongs to the group of genes that control the litter size of the sows and thus BF gene could be used for Marker-assisted selection programmes for the genetic improvement of reproductive characteristics in livestock.

Avian β-defensins (AvβDs) constitute a family of antimicrobial peptides that are critical to innate immunity in chickens, providing protection against microbial pathogens including Salmonella Enteritidis (SE). As apart from the digestive tract another main route of SE colonization in birds is via infection of the oviduct and specifically of the vagina, the aim of this study was to investigate the expression of the complete family of AvβDs, in the chicken vagina in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the vaginal AvβDs expression. Expression analysis revealed that 11 members of the AvβD family were expressed in the chicken vagina. Quantitative real-time PCR analysis revealed that the mRNA abundance of five AvβDs was up regulated and of one AvβD was down regulated with respect to sexual maturation. In addition SE infection resulted in a significant induction of AvβD5, 7, 10, 11, 12 and 14 in the vagina of sexually mature birds, and in a significant induction of AvβD5 and 11 in the vagina of aged birds. These findings provide strong evidence to suggest that an AvβD-mediated immune response mechanism exists in the chicken vagina providing protection against bacterial pathogens including Salmonella species.