小尾寒羊五个微卫星基因座遗传多态性研究

小尾寒羊是我国优良的地方绵羊品种,具有极高的繁殖力,平均每胎产羔2．6只。利用与绵羊高繁殖力主效基因Fec^B和FecX^1连锁的5个微卫星标记（OarAE101,BM1329,BMS2508,TGLA54t TGLA68)对244小尾寒羊母羊进行了遗传检测。用非变性（中性）聚丙烯酰胺凝胶电泳检测同卫星的PCR扩增产物,计算了5个同卫星基因座的等位基因频率,多态信息含量,基因纯合度和杂合度。在小尾寒羊中检测到BM1329有6个等位基因,片段大小为160－180bp,164bp等位基因频率最高（0．6320）;检测到OarAE101有9个等位基因,片段大小为97－135bp,97bp等位基因频率最高（0．7930）;检测到TGLA54有5个等位基因,片段大小为116－136bp,134bp等位基因频率最高（0．8500）;检测到TGLA68有2个等位基因,片段大小为98－100bp,2个等位基因频率相近,检测到BMS2508有6个等位基因,片段大小为93－115bp,99bp等位基因频率最高（0．4795）。BM1329,OarAE101,TGLA54,TGLA68,BMS2508的多态信息含量／基因纯合度／杂合度分别为0．4481／0．4840．0．5160,0．3516／0．6375／0．3625,0．2528／0．7326／0．2674,0．3733／0．5034／0．4966,0．5809／0．3581／0．6419。可见BMS2508的遗传变异最大,TGLA54的遗传变异最小。这些结果可为小尾寒羊种质特性研究提供分子基础数据。     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Effect of the rumen ciliates Entodinium caudatum,Epidinium ecaudatum and Eudiplodinium maggii,and combinations thereof,on ruminal fermentation and total tract digestion in sheep

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 × 2 × 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH₄-to-CO₂ ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.     

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesis cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes ofSalmonella typhimurium, cysE,cysM andcysK, and linkedcysEM,cysME andcysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and-cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and-cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cellsin vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.     

Genetic selection, sex and feeding treatment affect the whole-body chemical composition of sheep

Hypotheses on total body chemical composition were tested using data from 350 Suffolk sheep grown to a wide range of live weights, and fed in a non-limiting way, or with reduced amounts of feed, or ad libitum on feeds of reduced protein content. The sheep were from an experiment where selection used an index designed to increase the lean deposition rate while restricting the fat deposition rate. Ultrasound muscle and fat depths were the only composition measurements in the index. The animals were males and females from a selection (S) line and its unselected control (C). The protein content of the lipid-free dry matter was unaffected by live weight, sex or feeding treatment with only a very small effect of genetic line (0.762 kg/kg in S and 0.753 kg/kg in C; P < 0.05). The form of the relationship between water and protein was not affected by any of the factors; in the different kinds of sheep it was consistent with no effect other than through differences in mature protein weight. The water : protein ratio at maturity was estimated as 3.45. Over the whole dataset, lipid weight (L) increased with protein weight (P) according to L = 0.3135 × P1.850. Allowing for this scaling, fatness increased on low-protein feeds, was greater in females than in males and in C than in S (P < 0.001). Lipid content (g/kg fleece-free empty body weight) was reduced by restricted feeding only in males at the highest slaughter weight (114 kg). The lines differed in lipid content (P < 0.001) with means of 265.1 g/kg for C and 237.3 g/kg for S. Importantly, there was no interaction between line and feeding treatments. A higher proportion of total body protein was in the carcass in S than in C (0.627 v. 0.610; P < 0.001). For lipid, the difference was reversed (0.736 v. 0.744; P < 0.05). The total energy content increased quadratically with slaughter weight. At a particular weight, the energy content of gain was higher in females than in males and in C than in S. Genetic selection affected body composition at a weight favouring the distribution of protein to the carcass and lipid to the non-carcass. Once allowing for effects of genetic selection, sex and feeding treatment on fatness, simple rules can be used to generate the chemical composition of sheep.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.     

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes. Received: 14 February 2000 / Accepted: 16 August 2000     

Comparing the diversity of the casein genes in the Asian mouflon and domestic sheep

We aimed to determine whether casein variants that are currently segregating in ovine populations existed before the domestication of sheep or, to the contrary, if their emergence is much more recent. To this end, we have retrieved whole-genome sequences from Iranian and domestic sheep from Africa, Europe, South and East Asia and West Asia. Population structure analysis based on 55,352,935 SNPs revealed a clear separation between Iranian mouflons and domestic sheep. Moreover, we also observed a strong genetic differentiation between Iranian mouflons sampled in geographic areas close to Tehran and Tabriz. Based on sequence data, hundreds of SNPs mapping to the casein α_S1 (CSN1S1, 248 SNPs), casein α_S2 (CSN1S2, 268 SNPs), casein ß (CSN2, 146 SNPs) and casein κ (CSN3, 112 SNPs) genes were identified. Approximately 25–63.02% of the casein variation was shared between Iranian mouflons and domestic sheep, and the four domestic sheep populations also shared 44.2–57.4% of the casein polymorphic sites. These findings suggest that an important fraction of the casein variation present in domestic sheep was already segregating in the mouflon prior to its domestication. Genomic studies performed in horses and dogs are consistent with this view, suggesting that much of the diversity that we currently detect in domestic animals comes from standing variation already segregating in their wild ancestors.     

湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (−12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.     

Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity

Normal spermatogenesis is heavily dependent on the balance of germ cell proliferation, differentiation and apoptosis. Growth differentiation factor 9 (GDF9) and cyclin-dependent kinase inhibitor 1 B (CDKN1B) are strongly associated with cell cycle transition from G0/G1 to S and G2/M phase and hence regulating the growth and development of testicular germ cells and somatic cells. The current study was aimed at seeking out scientific evidence to determine if GDF9 and CDKN1B gene expression functions in the development of Tibetan sheep testes. To this end, developmental testes were derived from three-month-old (pre-puberty), one-year-old (sexual maturity), and three-year-old (adult) Tibetan sheep and then the expression and localization patterns of GDF9 and CDKN1B in these testes were evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunofluorescence. qRT-PCR and Western blot results showed that GDF9 and CDKN1B were detected in the testes throughout the different developmental stages. The abundance of GDF9 mRNA and protein in the testes of one- and three-year-old Tibetan sheep were higher than that in the testes of three-month-old Tibetan sheep; the mRNA and protein abundance of the CDKN1B gene in three-month-old Tibetan sheep testes were higher than that in the testes of the one-and three-year-old sheep. Moreover, immunofluorescence results suggested that the GDF9 protein was expressed in spermatogonia and Leydig cells, and that the CDKN1B protein was localized mainly in Leydig cells with some in the seminiferous epithelium throughout developmental stages. This indicated a novel role of the GDF9 and CDKN1B genes in Leydig cell development over and above their known roles in germ cell development. These findings have significant implications for our understanding of the molecular mechanisms of GDF9 and CDKN1B genes in Tibetan sheep spermatogenesis.     

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin, lactation and dry periods. A total of 122 genes were identified as differentially expressed, including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods. Gene ontology analysis showed the functional classification of the up-regulated genes in lactation, including transport, biosynthetic process, signal transduction, catalytic activity, immune system process, cell death, and positive regulation of the developmental process. Microarray data clarified molecular events in bovine mammary gland lactation.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Effects of sampling location and time,and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen

Aims: To investigate, using culture‐independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) profiles were similar among digesta samples from five locations (95·4%) and three time points (93·4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85·5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.     

Nitrogen excretion of adult sheep fed silages made of a mixed sward or of pure unfertilised grass alone and in combination with barley

Four adult rumen-fistulated wether sheep were fed silages combined with barley. The silages consisted of 48% grasses, 28% legumes and 24% other forbs (GCF) or of pure grass (G). The swards received no mineral fertiliser. The dry matter (DM) and fibre contents were lower in GCF than in G. Crude protein content of DM in GCF and G were 145 g/kg and 102 g/kg respectively. DM content as ash, lipids and non-fibrous carbohydrates were rather similar in both silages. About 40g DM were offered per kg BW^0.75 and day either as silage alone or as a mixture of silage and barley (60:40). Faecal N excretion was greater with GCF than with G. The proportion of faecal bacterial and endogenous debris N reached 75 and 73% when GCF or G was fed, respectively. Undigested dietary N represented about 20%, and water soluble N accounted for 5–6% of faecal N. GCF caused more urinary N than G. Barley reduced urinary N excretion when supplemented to GCF. No dietary influence on urinary non-urea nitrogenous compounds was shown. GCF caused higher urinary urea N excretion than G and barley reduced this fraction when replacing part of GCF. Based on the urinary urea N proportions, it is concluded that N intake exceeded N requirement for any of the four diets fed. Dietary supplementation of ruminally fermentable carbohydrates can reduce urinary N excretion and this improves the efficiency of utilisation of N in N-unfertilised biodiverse grassland/ruminant farming systems.