Effects of maternal treatment with β‐hydroxy‐β‐metylbutyrate and 2‐oxoglutaric acid on femur development in offspring of minks of the standard dark brown type

The aim of the study was to evaluate the effect of the diet, mother type and sex of the offspring on the mechanical and geometric parameters of long bones as well as bone tissue density in minks. Primiparous and multiparous dams were supplemented with β‐hydroxy β‐methylbutyrate (a metabolite of leucine, at the daily dosage of 0.02 g/kg of body weight) and/or 2‐oxoglutaric acid (a precursor of glutamine, at the daily dosage of 0.4 g/kg of body weight) during gestation. The diet did not influence bone tissue density and the length of the humerus. An increase in the length of the femur was noted in male offspring delivered by multiparous dams. The diet resulted in an increase in the weight of the humerus in males from multiparous dams and a decrease in offspring from primiparous dams. Heavier femora were noted in male offspring delivered by both types of dams. The maximum elastic strength of the humerus was higher in the offspring delivered by multiparous than primiparous dams, irrespective of the offspring sex. The diet resulted in reduction in the ultimate strength of the femur in the male offspring delivered by primiparous dams. Only females born by multiparous dams, irrespective of the diet, showed a significant increase in the cross‐sectional area of the humerus, while a significant decline was noted in males delivered by multiparous dams and in all the offspring delivered by primiparous dams. An increase in the cross‐sectional area of the femur was noted in the offspring delivered by multiparous dams, while reduction was observed in the offspring delivered by primiparous dams. These results have shown for the first time that the presence of β‐hydroxy‐β‐methylbutyrate or 2‐oxoglutaric acid in the diet of pregnant primiparous or multiparous dams unambiguously affects the geometry and mechanical properties of offspring's long bones.     

Dietary supplementation of β‐hydroxy‐β‐methylbutyrate in animals – a review

The leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) has been studied by many researchers over the last two decades. In particular, the utility of HMB supplementation in animals has been shown in numerous studies, which have demonstrated enhanced body weight gain and carcass yield in slaughter animals; positive immunostimulatory effect; decreased mortality; attenuation of sarcopenia in elderly animals; and potential use in pathological conditions such as glucocorticoid‐induced muscle loss. The aim of this study was to summarize the body of research on HMB supplementation in animals and to examine possible mechanisms of HMB action. Furthermore, while the safety of HMB supplementation in animals is well documented, studies demonstrating efficacy are less clear. The possible reasons for differences in these findings will also be examined.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

The high prolificacy of D’man sheep is associated with the segregation of the FecLL mutation in the B4GALNT2 gene

Mutations in the FecL locus are associated with large variation in ovulation rate and litter size in the French Lacaune sheep breed. It has been shown that the B4GALNT2 gene within the FecL locus is most likely responsible for the high fecundity in the French breed. In this study, we have highlighted the segregation of the FecL^L mutation within the B4GALNT2 gene in North African sheep breeds and notably in the highly prolific D'man breed. Genotyping of a sample of 183 Tunisian D'man individuals revealed a high frequency (0.65) of the prolific allele FecL^Lwhich was attributed to the adoption of a decades‐old breeding strategy based on the selection of ewe lambs born from large litter size. Homozygous LL ewes showed a significantly increased litter size compared to heterozygous and non‐carrier ewes (FecL^L/FecL^L = 2.47 ± 0.09 vs. FecL^L/FecL⁺ = 2.23 ± 0.09, p < 0.05 and FecL⁺/FecL⁺ = 1.93 ± 0.18, p < 0.01). The presence of the FecL^Lpolymorphism in both D'man and Lacaune breeds argues for an ancestral origin of this mutation and brings an answer to the old question of the genetic determinism of the extreme prolificacy of the D'man ewes. The results of this study can help to establish planned genotype‐based mating allowing both higher profit for the breeders and an optimal management of the FecL^L mutation in D'man sheep populations.     

西农萨能奶山羊INHA基因启动子区多态性与产羔数和产奶量的相关分析

采用PCR-SSCP技术分析了INHA基因的启动子区2个基因座位在西农萨能奶山羊群体中的遗传多态性,结果在P1引物所扩增的目的片段发现遗传多态,有2种等位基因A和B,有3种基因型AA型、AB型、BB型;在P2引物所扩增的目的片段未发现遗传多态.对多态片段的测序分析表明:AA型与BB型相比在DNA序列的第506 bp处插入了一个碱基G,在第446 bp处碱基C→T.AB、BB基因型个体的平均产羔数显著高于AA型个体(P<0.05);AB、BB基因型的个体各胎次产奶量均比AA型高,其中在第2、3胎产奶量和前4胎平均产奶量都达到显著水平(P<0.05),AB基因型的前4胎平均产奶量和AA型差异极显著(P<0.01).结果表明INHA基因对奶山羊产羔数和产奶量都有显著影响,因此认为INHA基因的P1位点可作为奶山羊的有效分子标记.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

β‐carotene attenuates lipopolysaccharide‐induced inflammation via inhibition of the NF‐κB,JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages

β‐carotene is one of the most abundant carotenoids, has potential anti‐inflammatory effect, it has been reported that β‐carotene could suppress LPS‐induced inflammatory responses by inhibiting nuclear factor kappa B (NF‐κB) translocation, but the more detailed molecular mechanisms underlying the anti‐inflammatory action of β‐carotene remain to be fully understood. In this study, we investigated the influence of β‐carotene on the activation of JAK2/STAT3, MAPK, and NF‐κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β‐carotene for 3 hr after LPS treatment for 24 hr. The mRNA expression and the release of IL‐1β, IL‐6, and TNF‐α were evaluated by RT‐PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF‐κB signaling pathway were detected by Western blot. The results showed that β‐carotene significantly suppressed (p < 0.05) LPS‐induced release of IL‐1β, IL‐6, and TNF‐α and their mRNA expression. LPS‐induced JAK2/STAT3, IκB/NF‐κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p < 0.05) by β‐carotene in a dose‐dependent manner. In conclusion, β‐carotene could attenuate LPS‐induced inflammation via inhibition of the NF‐κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.     

Effects of β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin in β‐carotene‐deficient Japanese Black cows

Data from 18 β‐carotene‐deficient Japanese Black cows were collected to clarify the effects of feeding β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin (Ig) in cows. Cows were assigned to control or carrot groups from 3 weeks before the expected calving date to parturition, and supplemental β‐carotene from dry carrots was 138 mg/day in the carrot group. Plasma β‐carotene concentrations in the control and carrot groups at parturition were 95 and 120 μg/dL, and feeding dry carrots slightly improved plasma β‐carotene at parturition. Feeding dry carrots increased colostral IgA concentrations in cows and tended to increase colostral IgG₁, but colostral IgM, IgG₂, β‐carotene and vitamin A were not affected by the treatment. Feeding dry carrots had no effects on plasma IgG₁, IgA and IgM concentrations in cows, but plasma IgG₁ concentrations decreased rapidly from 3 weeks before the expected calving date to parturition. These results indicate that feeding β‐carotene‐enriched dry carrots is effective to enhance colostral IgA and IgG₁ concentrations in β‐carotene‐deficient cows.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

ORIGINAL ARTICLE: Putative reference genes for gene expression studies in propionate and β‐hydroxybutyrate treated bovine adipose tissue explants

Accurate gene expression normalization using a stable reference gene (RG) improves the reliability of quantitative real‐time PCR (qPCR) results. Therefore, a validation of RGs should be done before their use. Only few studies on RGs have been done in cattle, and none in bovine adipose tissue (AT) explants, therefore, we characterize the effects of an in vitro treatment with propionate and β‐hydroxybutyric acid (BHB) on the mRNA content of these RGs comparing the output data from the geNorm^TM and the Normfinder^© program. geNorm^TM and Normfinder^© estimated the most stable RGs in the following sequence for subcutaneous and for retroperitoneal AT explants treated with propionate: low density lipoprotein receptor‐related protein 10 (LRP10) > hippocalcin‐like 1 (HPCAL1) > glyceraldehyde‐phosphate‐dehydrogenase (GAPDH) > ribosomal protein S9 (RPS9) > RNA polymerase II (Pol II) > beta2 actin (ACTB) > 18S ribosomal RNA (18S rRNA). BHB treated AT explants yielded a different stability ranking for RGs using geNorm^TM: HPCAL1, GAPDH > Pol II > LRP10 > ACTB > RPS9 > 18S rRNA. Normfinder^© estimated a different stability ranking for the RGs as shown in the following sequence for subcutaneous and retroperitoneal AT explants treated with BHB: HPCAL1 > Pol II > GAPDH > ACTB > LRP10 > RPS9 > 18S rRNA. Subsequent pairwise analysis of variation of RGs using geNorm^TM suggested that LRP10, HPCAL1 and GAPDH should be used for accurate normalization of subcutaneous and retroperitoneal AT explants treated with propionate, while HPCAL1, GAPDH and Pol II should be used for BHB treatment.     

崇明白山羊gdf-9基因多态性与产仔率的关联分析

上海崇明白山羊具有高繁殖率的优良特性,为探索这一特性的遗传基础,本研究利用PCR-SSCP联合PCR产物测序的方法分析了gdf-9基因的多态性,结果发现在崇明白山羊gdf-9基因的编码区内存在3处多态位点,分别为A183C、G1188A和G1189A。其中G1188A变异位点在其他山羊中尚未有报道。经氨基酸编码分析,A183C和G1188A处的多态性未造成氨基酸的编码改变,G1189A处的G→A多态变化导致了缬氨酸→异亮氨酸的改变。两相邻多态位点G1188A和G1189A的产仔率分析结果表明:在75只崇明白山羊中,G1188A位点的3种等位基因型G(G)、G(A)和A(A)的基因频率分别为86.67%、13.33%和0;G1189A位点的3种等位基因型G(G)、G(A)和A(A)的基因频率分别为60.00%、40.00%和0;G1188A位点处G(G)型个体产仔数的最小二乘均值大于G(A)型个体(P>0.05);G1189A位点G(A)型个体的平均产仔数大于G(G)型个体(P>0.05)。综合G1188A和G1189A两个位点,不同基因型的最小二乘均值关系为AC>AA>AB,但3种基因型间差异不显著(P>0.05)。综上结果,崇明山羊gdf-9基因变异位点的多态性对产仔数没有显著性影响。     

Thiamine deficiency affects glucose transport and β‐oxidation in rats

Thiamine is recognized as a cofactor for many enzymes involved in intermediary metabolism responsible for energy production. Animal model of thiamine deficiency (TD) included direct evaluation of glucose uptake by estimation of ³H‐deoxyglucose transport across red blood cells membranes and β‐oxidation of fatty acids in isolated leucocytes. Feeding of animals with the thiamine‐deficient diet (0.018 mg/kg diet) for 30 days resulted in disturbances in energy production. The thiamine intake was limited not only by vitamin B₁ deficiency in the diet, but also by time‐dependent drop of feed consumption by rats fed this diet. At the end of experiment, diet consumption in this group of rats was 52% lower than in the control group. This was accompanied by low glucose uptake by erythrocytes of rats suffering vitamin B₁ deficiency for longer time. At the end of experimental period, glucose uptake was over 2 times lower in TD erythrocytes than in control RBC. Such drop of energy production was not compensated by delivery of energy from fatty acid degradation. In leucocytes from TD rats, the β‐oxidation was also suppressed. Observed significant decrease of serum insulin from 2.25 ± 0.25 ng/ml (day 0) to 1.94 ± 0.17 ng/ml (day 30) might have significant impact on observed energy production disorders. The results from this study indicate that the thiamine deficiency significantly reduces feed intake and causes modest abnormalities in glucose and fatty acid utilization.     

Effects of supplemental β‐carotene on colostral immunoglobulin and plasma β‐carotene and immunoglobulin in Japanese Black cows

Data from 26 Japanese Black cows were collected to clarify the effects of supplemental β‐carotene on colostral immunoglobulin (Ig) and plasma β‐carotene and Ig in the cows. Cows were assigned to control or β‐carotene groups from 21 days before the expected calving date to 60 days after parturition. Supplemental β‐carotene was provided at 500 mg/day in the β‐carotene group. Supplemental β‐carotene drastically increased plasma β‐carotene concentrations in the cows from parturition to 60 days after parturition, and plasma β‐carotene concentrations in the control and β‐carotene groups at parturition were 202 and 452 μg/dl, respectively. Supplemental β‐carotene had no effects on plasma IgG₁, IgA or IgM concentrations at parturition. Supplemental β‐carotene increased colostral IgG₁ concentrations in the cows, but colostral β‐carotene, IgA and IgM concentrations were not affected by supplemental β‐carotene. These results indicate that supplemental β‐carotene is effective to enhance colostral IgG₁ concentrations and plasma β‐carotene concentrations in Japanese Black cows.     

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/β‐catenin signalling pathway in the Angora rabbit

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene β‐catenin and lymphoid enhancer‐binding factor 1 (LEF1) in Wnt/β‐catenin pathway. The levels of β‐catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of β‐catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle‐regulatory genes, insulin‐like growth factor‐1 (IGF‐1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/β‐catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/β‐catenin signalling pathway that promotes the proliferation of DP cells.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

DNA polymorphism of 5' flanking region of prolactin gene and its association with litter size in sheep

A single nucleotide polymorphism of 5' flanking region of the prolactin gene was investigated in both high prolificacy breeds (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset and Suffolk sheep) using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). The results indicated that two genotypes (AA and AB) were detected in Small Tail Han sheep (n   =   239), only one genotype (AA) was detected in Hu (n   =   40), Dorset (n   =   50) and Suffolk sheep (n   =   39). The mutant homozygous genotype (BB) was not detected in four sheep breeds. In Small Tail Han sheep (n   =   239), the frequency of genotypes AA and AB was 0.91 and 0.09, the frequency of the A and B alleles was 0.95 and 0.05, respectively. The fitness tests showed that the Small Tail Han sheep population was in Hardy–Weinberg equilibrium. Sequencing revealed a mutation (G→T) at the position 63 bp of the 5' flanking region of prolactin gene in AB genotype compared with AA genotype in Small Tail Han sheep. The Small Tail Han ewes with AB genotype had 0.83 (p < 0.05) lambs more than those with AA genotype. These results preliminarily showed that the prolactin locus is either a major gene that influences the high prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

Effect of the β‐carotene oxygenase 2 genotype on the content of carotenoids,retinol and α‐tocopherol in the liver,fat and milk of rabbit does,reproduction parameters and kitten growth

Mutations in the β‐carotene oxygenase 2 (BCO2) gene can impair the function of the enzyme that breaks down carotenoids. As a result, gradual accumulation of unoxidized carotenoids in animal tissues gives them a yellow colour. The aim of the study was to determine the content of carotenoids, retinol and α‐tocopherol in the liver, fat and milk of rabbit does with three different genotypes determined by AAT‐deletion mutation at codon 248 of the BCO2 gene and to find out whether differences in the concentrations of the above compounds in the tissues and milk of the does affect reproduction parameters and the rearing rate of kittens. The experimental materials comprised 36 does, 12 of each genotype of the BCO2 gene, with their litters. Females with their litters were placed in individual cages, on deep litter. Between days 7 and 13 of lactation, samples of milk were collected from the does. The kittens stayed with their mothers until 35 days of age. After weaning, the does were sacrificed. Tissue samples of liver and perirenal fat were collected for chemical analyses. Additionally, based on samples taken from one female, RNA expression levels were determined from the mammary gland and liver, adipose tissue and skin. It was found that homozygous does with deletion at codon 248 of the BCO2 gene were characterized by considerably higher concentrations of xanthophylls and beta‐carotene in the liver, adipose tissue and milk than does with the remaining genotypes. However, the differences in the content of the above compounds in milk had no influence on litter weight or the number and rearing rate of kittens. Additionally, RNA expression of the BCO2 gene was found in the mammary tissue of lactating doe and its level was similar to those noted in the liver and adipose tissue.     

Polymorphism of GDF9 and BMPR1B genes and their association with litter size in Markhoz goats

The main objective of this study was to investigate the polymorphism of GDF9 and BMPR1B genes and their relationship with litter size in Markhoz goats. The polymorphism of GDF9 and BMPR1B genes as well‐documented genes regarding fecundity in sheep and goat was investigated using RFLP‐PCR and a tetra‐primer amplification refractory mutation system‐PCR (T‐ARMS‐PCR) in Markhoz goats. The 164 blood samples were collected from the raised goats in Sanandaj Markhoz goat Performance Testing Station. The DNA extraction was carried out by salting‐out procedure, and then, PCR was performed using four and two pairs of primers to detect polymorphism in GDF9 and BMPR1B genes, respectively. To disclose GDF9 loci polymorphism, PCR products were digested with SspI (G3288A), PvuII (G423A), MvaI (A959C) and MspI (G1189A) restriction enzymes. The results showed that these mutations are available in tested animals. Parity had no significant effect on litter size. Also, the effects of different genotypes of GDF9 and BMPR1B had no significant effect on litter size. Further studies with a high number of animals with minimum relatedness for testing the association of these SNPs and others in the fecundity genes with reproductive traits may be worthwhile.