M. Avdi

Transcriptional profiling of Toll-like receptors in chicken embryos and in the ovary during sexual maturation and in response to Salmonella enteritidis infection

One of the key members of the innate immune system in many vertebrate species is the family of Toll-like receptors (TLRs). These molecules, which initiate the innate immune response and mount an anti-microbial response in both vertebrates and invertebrates, have recently been identified in the chicken genome. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS) infection are very important for reproductive physiology due to the transovarian transmission of Salmonella enteritidis (SE) in laying hens. The aim of this study was to investigate the expression of the ten cTLRs identified to date, in the chicken ovary in vivo and embryos during early embryonic development, to investigate whether sexual maturation affects their ovarian mRNA abundance and to investigate the transcriptional changes of TLRs in the chicken ovary in response to SE infection. RNA was extracted from embryos from day 3 to day 10 of embryonic development as well as from the ovaries of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all TLRs apart from TLRs 1-1 and 2-2 were expressed in the ovary of sexually mature chickens, while all TLRs apart from TLR1-1 were expressed in the chicken embryos during embryonic development. Quantitative real-time PCR analysis revealed that the ovarian mRNA abundance of TLRs differ with respect to sexual maturation. SE infection resulted in a significant induction of TLR4, and 15 in the ovary of sexual mature birds, and in a significant induction of TLR15 in the ovary of aged birds, while a significant down-regulation was observed for TLR3 in the ovary of aged birds. These findings suggest that a TLR-mediated immune response mechanism exists in the chicken ovary.

Transcriptional profiling of antimicrobial peptides avian β-defensins in the chicken ovary during sexual maturation and in response to Salmonella enteritidis infection.

Avian β-defensins (AvβDs) are antimicrobial peptides that play significant roles in the innate immune system in chickens. The aim of this study was to identify the types of AvβDs expressed in the chicken ovary, to investigate the effects of sexual maturation in the ovarian mRNA abundance and to determine the changes in their expression levels as a result to Salmonella enteritidis (SE) infection. RNA was extracted from the ovary of healthy prepubertal, sexually mature and aged birds, as well as from sexually mature and aged SE infected birds. Real-time PCR analysis revealed that 11 AvβDs genes were expressed in the chicken ovary. A significant up regulation of AvβD1, 3, 4, 5, 7 and 11 was observed in the ovary of sexually mature and aged birds. Furthermore, a significant up-regulation of AvβD4, 5, 7, 11 and 12 was observed in the ovary of SE infected sexually mature birds. These results suggest that the mRNA expression of at least six AvβDs increase with age in the ovary of laying hens, and that at least five AvβDs show an induction in their expression in response to SE infection, indicating an AvβD-mediated immune response mechanism in the chicken ovary.

Effects of sexual maturation and Salmonella infection on the expression of Toll-like receptors in the chicken vagina.

Toll-like receptors (TLRs) are a critical component of the innate immune response in many vertebrates, including avian species. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles and in the chicken ovary in vivo, as well as the changes in their expression in response to lipopolysaccharide or Salmonella enteritidis (SE) infection, have broad implications for reproductive physiology and for the prevention of transmission of zoonotic diseases to humans through the consumption of contaminated poultry eggs. Because the main route of egg contamination is from infection of the oviduct and mainly from the vagina, the aim of this study was to investigate the expression of the ten cTLRs identified to date in the chicken oviduct in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the expression of TLRs in the chicken vagina. RNA was extracted from the vagina of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all types of cTLRs apart from TLR1-1 were expressed in the vagina of sexually mature birds. Quantitative real-time PCR analysis revealed that the mRNA abundance of TLR2-1, 2-2 and 4 differ with respect to sexual maturation in the chicken vagina. SE infection resulted in a significant induction of TLR5 and 15 in the vagina of sexually mature birds, and in a significant induction of TLR2-1, 4 and 15 in the vagina of aged birds, while a significant down-regulation was observed for TLR7 in the vagina of sexually mature birds. These findings suggest that a TLR mediated immune response mechanism exists in the chicken vagina, playing a crucial role in preventing microbial pathogens from being incorporated into newly forming eggs.

Variations saisonnières du comportement d'œstrus et de l'activité ovulatoire chez les brebis Chios et Serres en Grèce

Sheep are seasonal breeders, but a lower seasonality has been reported for sheep breeds from the Mediterranean countries enabling spring matings. To further substantiate this, the present study compared seasonal variation in oestrus behaviour and ovulation in two Greek breeds (Chios and Serres) maintained in the same environment. In 27 adult ewes of each breed, oestrus behaviour, the occurrence of ovulation and ovulation rate were monitored during 2 successive years by daily inspection by rams, weekly progesterone assay in circulating blood plasma and monthly ovarian laparoscopy. The periods of anoestrus (109 +/- 8 (wk) vs 178 +/- 5 d) and anovulation (63 +/- 8 vs 149 +/- 6 d) were significantly shorter (P < 0.001 and P < 0.001) and more variable in Chios than in Serres ewes. Furthermore, the percentage of ewes exhibiting oestrus at least once monthly was significantly higher from March to July in Chios than in Serres ewes. Continuous ovulation throughout the year was noted in 5 and 2 Chios ewes during year 1 and 2, respectively. Ovulation rate (3.25 +/- 0.15 vs 1.45 +/- 0.04) was higher in Chios than in Serres ewes (P < 0.001) while duration of oestrus was also longer in Chios ewes (1.8 +/- 0.05 vs 1.5 +/- 0.06 d; P = 0.002). There were significant correlations between the duration of anovulatory periods of the 2 successive years in Chios (r = 0.43) but not Serres ewes. Ovulation rate was positively related to the onset of the sexual season in Serres but not in Chios ewes. The present data suggest that: i) the efficiency of Serres but not in Chios ewes. The present data suggest that: i) the efficiency of spring mating in these breeds is suboptimal since it is a period when the percentage of ewes exhibiting oestrus and ovulation as well as ovulation rate are limited, ii) owing to the large variation in seasonality within Chios ewes and to the good between year correlation in features of seasonality, selection against seasonality in Chios ewes could be worthwhile.

Changes in the expression of Toll-like receptors in the chicken testis during sexual maturation and Salmonella infection

Rooster infertility is a major concern in the poultry industry and chicken male reproductive organs are the infectious tissues of various pathogenic microorganisms. Protection of the chicken male reproductive organs from pathogens is therefore an essential aspect of reproductive physiology. Recently Toll-like receptors (TLRs) have been identified as one of the key components of innate immunity in vertebrate species and have been reported to be expressed in the reproductive organs in various female species, including the chicken. However, mechanisms of antimicrobial protection of male reproductive organs mediated by TLRs are poorly understood. The objectives of this study were to determine the expression profile of the entire family of the ten chicken TLR genes in the chicken testis, to investigate whether sexual maturation affects their testicular mRNA abundance and to determine the changes in their expression levels in response to Salmonella enteritidis (SE) infection. RNA was extracted from the testis of healthy pre-pubertal, sexually mature and aged birds, and from sexually mature SE infected birds. RT-PCR analysis revealed that all TLRs, apart from TLR1-1 (TLR6), were expressed in the chicken testis. Quantitative real-time PCR analysis revealed that the testicular mRNA abundance of certain TLRs was developmentally regulated with respect to sexual maturation, while SE infection resulted in a significant induction of TLR2-1, 4, 5, 15 and 21 in the testis of sexually mature birds compared, to healthy birds of the same age. These findings provide strong evidence to suggest a key role of TLRs in the innate immune responses of chicken testis against Salmonella colonization.

Effect of polymorphisms at the STAT5A and FGF2 gene loci on reproduction, milk yield and lameness of Holstein cows

The objective of this study was to investigate the association of polymorphisms at the STAT5A and FGF2 gene loci with fertility, lactation milk yield and lameness in dairy cattle. Five hundred and eighteen primiparous Holstein cows were included in the study. Several reproductive traits were considered including conception rate (0/1) at first insemination, conception rate (0/1) in a 305-day lactation, number of inseminations per conception, interval (days) from calving to conception for cows that conceived in the first 305 days of lactation and age at first calving. Milk yield and lameness incidence were also recorded. Genotyping was performed using PCR-RFLP. The effect of allele substitution at each gene locus on reproductive traits, milk yield and lameness was assessed with single-trait mixed linear models. No significant associations were found between reproduction traits and any of the studied polymorphisms, apart from age at first calving, for which STAT5A polymorphism had a suggestive effect (P = 0.077). In addition, no significant effect of any polymorphism on lameness was found. Replacement of the C by G allele at the STAT5A locus was associated with a significant (P<0.05) increase in lactation milk yield, suggesting that this locus could be considered in gene assisted selection for the genetic improvement of milk production.

Temporal changes in the expression of avian β-defensins in the chicken vagina during sexual maturation and Salmonella infection

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.

Expression of chicken zygote arrest 1 (Zar1) and Zar1-like genes during sexual maturation and embryogenesis

Maternal mRNAs, which are expressed in oocytes, play an important role in the success of early embryo development, as they allow the first cleavages to occur. Zygote arrest 1 (Zar1) is an oocyte-specific maternal-effect gene that functions at the oocyte-to-embryo transition in many vertebrate species including human, pig, cattle, sheep, mouse, rat, frog and zebrafish. Recently, through in silico studies, a gene structurally related to Zar1, called Zar1-like has been identified in many vertebrates, including the chicken. The objectives of this study were to investigate the expression of the chicken Zar1 and Zar1-like genes in chicken tissues and embryos and to determine whether sexual maturation affects their mRNA abundance. RNA was extracted from various organs of chickens aged from one month up to two years old and from chicken embryos until day ten of embryonic development. Expression analysis of the genes was performed using RT-PCR and real-time PCR. RT-PCR analysis revealed that both genes were preferentially expressed in chicken oocytes, ovary and testes and in embryos during embryonic development. Quantitative real-time PCR analysis revealed a significant up regulation of Zar1 in the mature ovary, and also a significant up regulation of Zar1 and Zar1-like genes in the testes of sexually mature roosters, suggesting a key role of these genes in the chicken fertility. In contrast, expression of Zar1-like was not affected by age in the chicken ovary. Our results indicate that the chicken Zar1 and Zar1-like transcripts are co-expressed in high levels in the chicken gonads. In addition their expression beyond the stage of embryonic genome activation suggests an embryonic and not only a maternal origin of these transcripts.

Sources of variation and genetic profile of spontaneous, out-of-season ovulatory activity in the Chios sheep

Organising the breeding plan of a seasonally breeding species, such as sheep, presents a challenge to farmers and the industry as a whole, since both economical and biological considerations need to be carefully balanced. Understanding the breeding activity of individual animals becomes a prerequisite for a successful breeding program. This study set out to investigate the sources of variation and the genetic profile of the spontaneous, out-of-season ovulatory activity of ewes of the Chios dairy sheep breed in Greece. The definition of the trait was based on blood progesterone levels, measured before exposing the ewes to rams, which marks the onset of the usual breeding season. Data were 707 records, taken over two consecutive years, of 435 ewes kept at the Agricultural Research Station of Chalkidiki in northern Greece. When all available pedigree was included, the total number of animals involved was 1068. On average, 29% of all ewes exhibited spontaneous, out-of-season ovulatory activity, with no substantial variation between the years. Significant sources of systematic variation were the ewe age and live weight, and the month of previous lambing. Older, heavier ewes, that had lambed early the previous autumn, exhibited more frequent activity. Heritability estimates were 0.216 (± 0.084) with a linear and 0.291 with a threshold model. The latter better accounts for the categorical nature of the trait. The linear model repeatability was 0.230 (± 0.095). The results obtained in this study support the notion that spontaneous out-of-season ovulatory activity can be considered in the development of a breeding plan for the Chios sheep breed.

Expression of avian β-defensins and Toll-like receptor genes in the rooster epididymis during growth and Salmonella infection

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.