Prolactin Gene Research Articles

Polymorphisms of α-lactoalbumin, β-lactoglobulin and prolactin genes are highly associated with milk composition traits in Spanish Merino sheep

Traditionally, Merino Sheep has been used for wool and meat production. However, in Spain, it is used for manufacturing valuable artisanal cheeses. Therefore, milk production is becoming a very important trait to be considered. In this research, the effects of the different genetic variants of milk whey proteins α-lactoalbumin (LALBA) and β-lactoglobulin (LGB), and prolactin hormone (PRL) on milk quality traits have been estimated in 1114 lactations from 396 genotyped Merino ewes of an experimental flock. Nucleotide substitutions g.133C > T (A and B alleles) of LALBA gene and g.1617T > C (A and B alleles) of LGB gene and a deletion, g.460_483del, of PRL gene (A and B alleles) were analysed. Hardy-Weinberg Equilibrium (HWE) and fixation index (FIS) analyses detected that the genotyped Merino population have not been selected for the analyzed loci. Haplotype analyses of milk whey protein loci (LALBA and LGB) located at Ovis aries chromosome 3 (OAR3), indicate that alleles are independently inherited. So, we only studied the genetic effects of single genes on milk traits. Allele substitution and genotypes effects on milk yield and composition traits were estimated using a linear mixed model. Significant effects of LALBA *A (p. Ala27), LGB *A (p. Tyr38) and PRL *B (del24 bp) alleles on milk quality traits standardized to 120 days have been observed. LALBA *A allele is significantly associated with higher percentages of fat (FP; α = + 0.26%, P = 0.001), protein (PP; α = + 0.22%, P = 0.001), solids non fat (SNF; α = + 0.17%, P = 0.001) and total solids (TS; α = 0.42%, P = 0.0002). LGB *A allele shows significant positive effect on PP (α = + 0.136%, P = 0.032) and FP (α = + 0.140%, P = 0.070). PRL *B allele shows only a significant positive effect on PP (α = + 0.131%, P = 0.026). These positive effects could be of great interest for the cheese industry. Variants could be used as suitable markers to perform association analyses in commercial farms in order to implement a cheese yield selection program based on genotype information.

Inflammation and LPS-Binding Protein Enable the Stimulatory Effect of Endotoxin on Prolactin Secretion in the Ovine Anterior Pituitary: Ex Vivo Study.

Prolactin is a hormone that plays an important role in the regulation of many physiological processes including lactation, reproduction, fat metabolism, and immune response. The secretion of prolactin could be disturbed by an immune stress commonly accompanying infection. This study was designed to determine the influence of bacterial endotoxin—lipopolysaccharide (LPS)—on prolactin gene (PRL) expression and prolactin release from the ovine anterior pituitary (AP) explants collected from saline- and LPS-treated ewes in the follicular phase. The expressions of toll-like receptor 4 (TLR4) and proinflammatory cytokines interleukin- (IL-) 1β, IL-6, and tumor necrosis factor- (TNF-) α genes were also assayed. The results of the study showed that LPS stimulates prolactin secretion and IL-6 gene expression in the AP explants, but its action on lactotrophs depends on the immunological status of animal. It was demonstrated that an important role in enhancing the effect of LPS on the pituitary in the saline-treated ewes is played by LPS-binding protein (LBP)- “adapter molecule” for LPS binding to the cell surface receptor CD14 and then to TLR4. Also, it was found that bacterial endotoxin acting on the anterior pituitary cells may enhance prolactin secretion, and this effect of LPS could be mediated by IL-6 which is known as prolactin-releasing factor. Identification of the neuroendocrine and immune interactions in the regulation of prolactin secretion could be helpful in developing newer and more effective treatments for dysfunctions connected with disorders in this hormone secretion.

Highly deleterious variations in COX1, CYTB, SCG5, FK2, PRL and PGF genes are the potential adaptation of the immigrated African ostrich population

Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.

Molecular analysis of milk yield and components associated with some genetic markers in dairy cattle.

Prolactin and Lactoferrine genes related with milk yield and milk components were analyzed for 180 milking cows. Genotyping for these two genes were investigated to detect their associations with yields of milk, fat and protein and age at first calving (AFC). About 38 local cows from Serw farm (Baladi) in Damietta governorate, 62 Friesian cows from Sakha farm and 80 from El Karada farm in Kafr El-Sheikh. Blood samples were collected from all cows in EDTA tubes and salting out method was used to extract DNA from these blood samples. PCR RFLP method was used to get single nucleotide polymorphism (SNP) by PCR technique. Three enzymes were used to restrict DNA product (HINF1 for lactoferrin gene, MRON1 and SML1 restriction enzymes for prolactin gene). There were significant differences among three genotypes (AA, AB and BB) detected by restriction enzyme HINF1 in Frisian cattle for milk, fat, protein yield traits but not significance for AFC trait. The genotype AB was the best one for milk, fat and protein yields. In local cattle at Serw farm, there was no significance differences among three genotypes for milk, fat and protein yield, but for age at first calving there were significance differences. The genotypes from restriction enzyme MRON1 were two genotypes (CD and DD) for El-Qarada herd. There were significant differences between the two genotypes in Frisian herd for milk, fat, protein yields, but significance for AFC trait. In Serw herd, there were not significant differences among the three genotypes for milk, fat and protein yields, but AFC was significance. Genotypes from restriction enzyme SML1 were GG, GT and TT. There were significance differences among three genotypes in Friesian herd for milk, fat and protein yields, while in AFC there were non-significance differences. In Sero herd, differences in genotyping among four traits were not significant.

Genetic diversity of Prolactin gene in Japanese quail (Coturnix Coturnix Japonica)

This study was carried out to investigate the genetic diversity of prolactin gene (PRL gene) in Japanese quails using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) marker. One hundred and thirty nine quails were sampled for PRL loci analysis. DNA was extracted from the samples. Polymerase chain reaction (PCR) and electrophoresis was used to characterize a 24 base pair (bp) insertion/deletion in a 358 bp PCR product. Test for Hardy-Weinberg Equilibrium were carried out. From the results, two alleles A (0.63) and B (0.37) and three genotypes AA, AB and BB were observed from this locus. Results of the study showed that genetic variability exists in the study population of Japanese quails in Nigeria and its segregation could be assessed for reproductive capacity

Prolactin regulatory element-binding (PREB) protein regulates hepatic glucose homeostasis

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin (PRL) gene expression. PRL, also known as luteotropic hormone or luteotropin, is well known for its role in producing milk. However, the role of PREB, in terms of hepatic glucose metabolism, is not well elucidated. Here, we observed expression of Preb in the mouse liver, in connection with glucose homeostasis. Morevoer, Preb was downregulated in db/db, ob/ob and high-fat diet-induced obese (DIO) mice, concurrent with upregulation of the liver genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase-1 (Pck). Administration of adenovirus-Preb (Ad-Preb) to db/db, ob/ob, and DIO mice diminished glucose, insulin, and pyruvate tolerance, which analogously, were impaired in normal (C57BL/6) mice knocked down for Preb, via infection with Ad-shPreb (anti-Preb RNA), indicating Preb to be a negative regulator of liver gluconeogenic genes. We further demonstrate that Preb negatively influences gluconeogenic gene expression, by directly binding to their promoters at a prolactin core-binding element (PCBE).A better understanding of Preb gene expression, during the pathogenesis of hepatic insulin resistance, could ultimately provide new avenues for therapies for metabolic syndrome, obesity, and type-2 diabetes mellitus, disorders whose worldwide incidences are increasing drastically.

Grass Carp Prolactin Gene: Structural Characterization and Signal Transduction for PACAP-induced Prolactin Promoter Activity

In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP38 which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.

Investigation of PRL-RsaI and HaeIII gene polymorphisms in Anatolian water buffaloes bred by using PCR-RFLP method

ABSTRACT The objective of this study was to investigate polymorphisms both in exons 1 and 3 of prolactin (PRL) gene for milk productivity of Anatolian water buffalo breed in Sivas province in Turkey. Blood samples were collected from 129 male and female water buffaloes and DNA was isolated by using phenol/chloroform method. Samples of DNA were amplified and resulting PCR products were digested with RsaI (for exons 1 and 3) and HaeIII (for exon 1). Allelic polymorphisms were determined by [...]

Polymorphism of PIT-1 and Prolactin Genes and Their Effects on Milk Yield in Holstein Frisian Dairy Cows Bred in Vietnam

The aim of this study is to investigate the polymorphism of the Pituitary transcription factor (PIT-1) and Prolactin (PRL) genes in Holstein Friesian cows (HF) bred in Vietnam and their effects on milk yields. For these purposes, 125 HF dairy cows of milking age from Da Lat Milk Farm located in Lam Dong, Vietnam were sampled. Phenotypic data of milk yields (calculated on 305-day of milking cycle; kg/day) of each individual milking cow were collected. The PIT-1.E2, PIT-1.E6, PRL.E3 and PRL.E4 genotypes were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with restriction enzymes TaqI, HinfI, RsaI, respectively. The significant effects of PIT-1.E6, PRL.E3 and PRL.E4 polymorphisms on milk yields were not observed in HF dairy cows (P > 0.05). However, results revealed statistically significant associations between PIT-1.E2 polymorphism and milk yields of HF dairy cows; individuals carrying the AA genotype yielded more milk compared to others. Our study suggests the potential use of PIT-1.E2 SNP in marker assisted selection (MAS) for improved milk yields of HF dairy cows bred in Vietnam.

Prolactin Gene and Laying Performance in Aseel Birds

Prolactin Gene and Laying Performance in Aseel Birds

Polymorphism of prolactin, growth differentiation factor 9, and calpastatin genes and their effects on weight traits in Awassi lambs.

Objectives:This study was conducted to determine the correlation among prolactin gene (PRG), growth differentiation factor 9 (GDF-9), and calpastatin (CAG) genes polymorphism with growth traits in Awassi lambs.Materials and Methods:Records of 779 Awassi lambs from 264 dams and 15 sires were used. The sex, type of birth (single versus twins), weight at birth, average daily gain (ADG), and the adjusted weight at 60 days of age were determined for each animal. Blood samples were collected from all lambs to determine PRG, GDF-9, and CAG polymorphism using polymerase chain reaction-Restriction fragment length polymorphism.Results:Three PRG genotypes (AA, AB, and BB) were determined with a frequency of 0.88, 0.05, and 0.07, respectively. The frequency of each allele was 0.84 and 0.16 for A and B alleles, respectively. For GDF-9, there were only two genotypes detected (AB and BB) with a frequency of 0.96 and 0.04, respectively. The frequency of each allele was 0.92 and 0.08 for A and B alleles, respectively. For CAG, two genotypes were also detected (AB and BB) with a frequency of 0.92 and 0.08, respectively. The frequency of each allele was 0.96 and 0.04 for A and B alleles, respectively. A significant (p ≤ 0.04) effect of PRG genotype on birth weight was detected but this effect was not significant on ADG and weight at weaning. There were no associations between any of the pre-weaning growth traits and GDF-9 and CAG variants.Conclusion:The results of this study show that PRG could be used to select dams with a high frequency of dystocia to reduce birth weight of newborn lambs and therefore conserve the dam’s reproductive functions and improve lamb survivability.

Adrenergic hormones induce extrapituitary prolactin gene expression in leukocytes-potential implications in obesity

The pituitary hormone prolactin (PRL), originally described for its role in lactation, has been implemented in over 300 functions and is produced by multiple cell types outside of the pituitary. Monocyte/macrophages in particular show robust expression of extra-pituitary prolactin (ePRL). While ePRL protein is identical to pituitary PRL and translated from the same gene, tissues outside the pituitary engage an alternative promoter to regulate expression. Many of the factors regulating this expression, however, remain unknown. Here we show that the adrenergic hormones epinephrine and norepinephrine induce PRL expression in the human monocytic cell line THP-1 at physiological concentrations. Furthermore, our experiments show the polarization state of differentiated macrophages can influence their response in vitro, with inflammatory M1 macrophages—common in obese adipose—showing the highest levels of PRL expression compared to other macrophage types. Adrenergic hormones have a clearly defined role in adipocyte lipid metabolism, stimulating lipolysis through hormone sensitive lipase (HSL) induction. Meanwhile, PRL has been shown to stimulate lipogenesis. This highlights ePRL production as a possible factor in obesity. The overall balance of these two signals could play a critical role in determining overall lipid turnover/accumulation in adipose depots where large numbers of adipose tissue macrophages (ATMs) reside.

Polymorphism of Prolactin Gene and Its Association with Egg Production Trait in Four Commercial Chicken Lines

Broodiness is a behavioral trait observed in most common breeds of domestic fowl and due to its fundamental role in avian reproduction, it has been of great interest to poultry scientists, breeders and producers of hatching eggs. Prolactin gene (PRL) is generally accepted as crucial to the onset and maintenance of broodiness in birds and thus plays a crucial role in egg production. Therefore, the present study aimed to screen the Single Nucleotides Polymorphisms (SNPs) of prolactin gene in four commercial chicken lines namely Hubbard F15, Lohmann, Cobb500, and Avian48 using PCR and direct sequencing. A total number of forty chickens (ten females from each of the four commercial chicken lines) were used. Blood samples were collected aseptically from brachial (wing) vein of the chickens for genomic DNA extraction. PCR reaction was done using five pairs of primers, one sense (F) and one antisense (R) primer for each of the five exons of prolactin gene. Finally, DNA sequencing and Single Nucleotide Polymorphisms (SNPs) analysis was done using Laser gene Megalign program. The results showed three SNPs in Hubbard F15 chicken line; one synonymous SNP at the position 3838 bp (ACC/ACT-transition) in exon 2 while in exon 5, two SNPs were detected; one non-synonymous single nucleotide polymorphism at the position 7921bp (CCT/TCT-transition) which results in amino acid changes at codon positions 169 (P/S), and one synonymous single nucleotide polymorphism at the position 8187 bp T/ C. The study concluded that this SNP in PRL gene could be used as the potential molecular markers for egg production traits in chicken.

Evolution of the growth hormone, prolactin, prolactin 2 and somatolactin family

Growth hormone (GH), prolactin (PRL), prolactin 2 (PRL2) and somatolactin (SL) belong to the same hormone family and have a wide repertoire of effects including development, osmoregulation, metabolism and stimulation of growth. Both the hormone and the receptor family have been proposed to have expanded by gene duplications in early vertebrate evolution. A key question is how hormone-receptor preferences have arisen among the duplicates. The first step to address this is to determine the time window for these duplications. Specifically, we aimed to see if duplications resulted from the two basal vertebrate tetraploidizations (1R and 2R). GH family genes from a broad range of vertebrate genomes were investigated using a combination of sequence-based phylogenetic analyses and comparisons of synteny. We conclude that the PRL and PRL2 genes arose from a common ancestor in 1R/2R, as shown by neighboring gene families. No other gene duplicates were preserved from these tetraploidization events. The ancestral genes that would give rise to GH and PRL/PRL2 arose from an earlier duplication; most likely a local gene duplication as they are syntenic in several species. Likewise, some evidence suggests that SL arose from a local duplication of an ancestral GH/SL gene in the same time window, explaining the lack of similarity in chromosomal neighbors to GH, PRL or PRL2. Thus, the basic triplet of ancestral GH, PRL/PRL2 and SL genes appear to be unexpectedly ancient. Following 1R/2R, only SL was duplicated in the teleost-specific tetraploidization 3R, resulting in SLa and SLb. These time windows contrast with our recent report that the corresponding receptor genes GHR and PRLR arose through a local duplication in jawed vertebrates and that both receptor genes duplicated further in 3R, which reveals a surprising asynchrony in hormone and receptor gene duplications.

Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells.

Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2'-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPβ- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPβ and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.

Expression profiles of immunity and reproductive genes during transition period in Holstein cattle.

The transition period is a critical time for dairy cows as the animal is subjected to the physiological stress accompanying parturition. Immunosuppression and health status were examined during this period in 80 Holstein cows. Blood samples were taken from each cow 3, 2 and 1week before and after calving, and at calving (0day). RNA was extracted and subjected to real-time PCR to determine mRNA levels for the immune-related genes TLR 2, 4, 6, 7 and β-defensin 5 in addition to the reproduction-related genes prolactin and IGF-I. Results showed significant up-regulation of pro-inflammatory-selected genes, TLR 4, 6 7 and β-defensin 5 at the third-week post-calving; however, earlier periods had lower expression of such genes. In contrast, the immunosuppression biomarker TLR2 gene was up-regulated at calving and 1week after parturition and then down-regulated again at second and third week. Prolactin and IGF-I genes expression levels were significantly and gradually increased mainly post-partum. This research highlights that the expression patterns of TLRs, BNBD5, PRL and IGF-I could be biomarkers to follow up immune and reproductive status of dairy cow at peri-parturient period to predict the most susceptible risk time for disease incidence and to build up management protocol.

An association analysis between PRL genotype and milk production traits in Italian Mediterranean river buffalo.

This Research Communication describes the association between genetic variation within the prolactin (PRL) gene and the milk production traits of Italian Mediterranean river buffalo (Bufala mediterranea Italiana). High resolution melting (HRM) techniques were developed for genotyping 465 buffaloes. The association of genetic polymorphism with milk production traits was performed and subsequently the effects of parity and calving season were evaluated. Single nucleotide polymorphisms (SNPs) were identified at exons 2 and 5 and at introns 1 and 2. All the SNPs were in Hardy-Weinberg equilibrium, and statistical analysis showed that the polymorphism of intron1 was significantly (P < 0·05) associated with milk yield, milk protein content and peak milk yield. The average contribution of the intron1 genotype (r 2 intron1) to total phenotypic variance in milk production traits was 0·09, and the TT genotype showed lower values than CC and CT genotypes. A nonsynonymous SNP was identified in exon 2, which resulted in an amino acid change from arginine to cysteine. Moreover, the polymorphism of exon 2 was associated significantly with milk fat content (P < 0·05), and the buffaloes with TT genotype showed higher total fat content than the buffaloes with CT genotype. These findings provide evidence that polymorphisms of the buffalo PRL gene are associated with milk production traits and PRL can be used as a candidate gene for marker-assisted selection in Italian Mediterranean river buffalo breeding.

Polymorphism of Exon V of prolactin gene and its association with Cashmere traits in Changthangi Pashmina goats

Prolactin is one of the potential genes which mediates seasonal cues entraining reproductive and hair follicle growth cycles. Seasonal changes in levels of prolactin hormone mediate the patterns of de-iodinase enzyme activity which influence the follicular activity and thereby may affect Cashmere fibre growth and moult. To study polymorphism of the prolactin gene and associate it with fibre production and quality, Changthangi goat 196 bp fragment of prolactin gene Exon-V region was amplified and restriction digested with ECO 241(BanII) and in the study three genotypes were observed. These different genotypes were confirmed by DNA sequencing. The frequencies for the observed genotypes A1A1, A2A2, A1A2 were 0.214, 0.291 and 0.495 respectively. The distribution of the genotypes within the Changthangi goat population studied herein did not deviate from the Hardy Weinberg equilibrium. The mean genetic variability parameters for the gene analyzed were 0.4970 (heterozygosity value), 0.3735 (PIC value), 1.9880 (Effective allele number) and 0.6901 (Shannon index I) . The polymorphism observed in the gene showed a non-significant association (P&gt;0.05) with Cashmere quality traits. The results obtained here in, because of low sample size, demand further investigation with sufficiently large number of samples.

Genetic variability of myostatin and prolactin genes in popular goat breeds in Egypt.

The genetic polymorphisms of two functional genes named: myostatin (MSTN) and prolactin (PRL) were investigated in three goat breeds (Barki, Damascus and Zaraibi) using Sanger nucleotide sequence and restriction fragment length polymorphism (RFLP) methods, in order to differentiate between these breeds. Nucleotide sequencing of 337 bp MSTN gene detected five SNPs in Barki breed, two SNPs in Damascus breed, while the Zaraibi breed did not show any SNPs. Moreover, MSTN-HaeIII/PCR-RFLP gave a single Genotype BB was found in all the studied breeds. Meanwhile, Nucleotide sequencing of 196 bp PRL gene showed two SNPs in Damascus breed, one SNPs in Zaraibi breed, while the Barki breed did not show any SNPs. Moreover, PRL-Eco24I/PCR-RFLP showed three genotypes (AA, AB and BB). The genotype AB showed the maximum frequency in all the studied breeds (0.75, 0.85, and 0.90 for Damascus, Barki and Zaraibi breeds, respectively). Observed heterozygosity (Ho) value was higher than expected heterozygosity (He) value all studied breeds. In addition, the values of both Ho and He were the highest in Zaraibi breed (0.90 and 0.51 respectively). Chi-square (χ2) value revealed a significant variation Hardy-Weinberg equilibrium (P < .05) in the three studied breeds. It is the highest in Zaraibi goats and lowest in Damascus breed. The results demonstrated that the PRL-Eco24I/PCR-RFLP polymorphism may be utilized as effective marker for genetic differentiation between goat breeds, but MSTN-HaeIII/PCR-RFLP revealed no polymorphism or variation, thus it is not recommended in the selection program. Moreover, these results open up interesting prospects for future selection programs, especially marker assisted selection. In addition, the results established that PCR-RFLP method is a suitable tool for calculating genetic variability.

Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector.

Bivalent vaccines based on live attenuated viruses expressing a heterologous protein are an attractive strategy to address co-infections with various pathogens in the field. Considering the excellent efficacy and safety of the lapinized live attenuated vaccine C-strain (HCLV strain) of classical swine fever virus (CSFV), we proposed that C-strain has the potential as a viral vector for developing bivalent vaccines. To this end, we generated three recombinant viruses based on C-strain, one expressing the capsid (Cap) gene of porcine circovirus type 2 (PCV2) with the nuclear localization signal (NLS) (rHCLV-2ACap), and the other two expressing the PCV2 Cap gene without the NLS yet containing the signal peptide of the prolactin gene (rHCLV-pspCap) or that of the ubiquitin-specific peptidase gene (rHCLV-uspCap). All the recombinant viruses exhibited phenotypes similar to those of the parental virus and produced high-level anti-CSFV neutralizing antibodies (NAbs) in rabbits. Interestingly, rHCLV-uspCap and rHCLV-pspCap, but not rHCLV-2ACap, elicited detectable anti-Cap and -PCV2 NAbs in rabbits. Taken together, our data demonstrate that C-strain can be used as a viral vector to develop bivalent vaccines.