Attenuation by leptin of the effects of fasting on ovarian function in hens (Gallus domesticus)

Thirty-four-week-old laying hens received injections of recombinant chicken leptin to assess the role of leptin in avian ovarian function. In the first experiment, the hens (n=60) were divided into three groups: (i). fed ad libitum; (ii). fasted; and (iii). fasted + leptin. Hens were fasted for 5 days and those treated with leptin received 250 microg leptin kg-1 body weight twice a day, i.p. In the second experiment, the hens (n=72) were divided into four groups: (i). fed ad libitum; (ii). fasted; (iii). fasted + leptin given only during fasting (5 days); or (iv). fasted and leptin given during both fasting and 5 days of re-feeding (10 days). LH was measured in blood plasma, and progesterone and oestradiol were measured in blood plasma and the ovary by radioimmunoassay. Apoptosis was examined in the walls of the three largest yellow hierarchical follicles (F3-F1; F38-12 mm), and the granulosa layer of F3 follicles. The expression of leptin receptor in the granulosa layer of F2 and F1 follicles was barely detectable. This was in contrast to a much higher expression of leptin receptor maintained in the theca layer of F3-F1 follicles. The present results indicate that in chickens leptin might be involved in the adaptation to starvation due to attenuation of follicular apoptosis. The presence of leptin receptors in the ovary indicates the possibility of a peripheral effect of the hormone.     

Changes in the localization of MHC class II positive cells in hen ovarian follicles during the processes of follicular growth, postovulatory regression and atresia

The aim of this study was to determine the changes in the population of major histocompatibility complex class II positive (MHC-II(+)) cells in ovarian follicles during the processes of follicular growth, postovulatory regression and follicular atresia in hens. Cryostat sections of ovarian stroma containing cortical follicles, small white follicles, the largest (F(1)) and third largest (F(3)) preovulatory follicles, postovulatory and atretic follicles of laying hens were prepared. The sections were immunostained for MHC-II molecules using mouse anti-chicken MHC-II monoclonal antibody and positive cells were counted using a computer-assisted image analyser under a light microscope. MHC-II(+) cells were localized in the theca layer of normally growing follicles including cortical follicles, small white follicles and F(3) and F(1) preovulatory follicles, whereas they were found in both the theca and granulosa layers in postovulatory and atretic follicles. The frequency of MHC-II(+) cells in the theca layer was significantly increased during follicular growth from cortical follicles to F(3) preovulatory follicles. Although the population of MHC-II(+) cells did not differ between F(3) and F(1) preovulatory follicles, it increased significantly in postovulatory follicles (P < 0.01). The population of MHC-II(+) cells was significantly greater in the theca layer of atretic follicles than in non-atretic follicles (P < 0.01). These results indicate that the antigen-presenting function via MHC-II increases in association with follicular growth. A marked increase in MHC-II(+) cells indicates that these cells may be involved in regression of postovulatory and atretic follicular tissues.     

Changes in the expression of interleukin-1beta and lipopolysaccharide-induced TNF factor in the oviduct of laying hens in response to artificial insemination

The aim of this study was to determine the physiological significance of interleukin-1beta (IL1B) and lipopolysaccharide-induced TNF factor (LITAF) in the fate of sperm in the oviduct of laying hens after artificial insemination (AI). Laying hens were inseminated with fresh semen, PBS or seminal plasma and tissues from different oviductal segments were collected to observe the general histology, changes in the mRNA expression of IL1B and LITAF and the localization of positive cells expressing immunoreactive IL1B (irIL1B). Semi-quantitative RT-PCR was used to observe the changes in mRNA expression of these molecules in the infundibulum, uterus, utero-vaginal junction (UVJ), and vagina after insemination. Intact sperm in the lumen and between the primary or secondary folds of the vagina were found until 6 h after insemination but were degraded at 12 h. The mRNA expression of IL1B and LITAF was significantly increased in the vagina until 6 h after AI but remained unchanged in the other oviductal segments. In the tissue of the vagina and UVJ, irIL1B was localized in the mucosal stroma. The number of irIL1B-positive cells was increased in the vagina but almost unchanged in UVJ after insemination with semen. Significant changes were not observed in the mRNA expression and irIL1B-positive cells in the vagina after PBS or seminal plasma insemination. The increase of IL1B and LITAF in the vagina may lead to sperm degradation and elimination by cilia of surface epithelium, whereas their lower levels in UVJ may permit sperm to survive in sperm storage tubules.     

Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.     

Changes in insulin-like growth factor binding protein (IGFBP) isoforms during bovine follicular development

UThe insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and so regulate their access to the type 1 and 2 IGF receptors. This is the principal mechanism involved in regulating IGF bioavailability during folliculogenesis. IGFBPs undergo a number of post-translational modifications, including proteolytic cleavage, phosphorylation and glycosylation, which can regulate the affinity of IGFBPs for IGFs. However, the post-translational changes to IGFBPs that occur during folliculogenesis have not been fully characterized. The charge and size variants of the IGFBPs in bovine follicular fluid were examined by two-dimensional non-reducing SDS-PAGE followed by non-isotopic western ligand blot analysis, and immunoblot analysis during follicular development. The results demonstrate the presence of at least 51 IGFBP isoforms corresponding to IGFBP-1 to -6 in bovine follicular fluid from subordinate follicles, many of which were phosphorylated. The total number of IGFBPs was reduced in dominant follicles, whereas no gross changes in isoforms were observed during follicular development. These results demonstrate the high degree of conservation of IGFBP post-translational modifications between species, and from the in vitro dephosphorylation of these proteins it is hypothesized that these modifications may result in changes to IGF binding or susceptibility to proteolytic cleavage.     

Seasonal effects on the response of ovarian follicles to IGF1 in mares

The response of follicles to IGF1 was compared between the transition into the ovulatory season (transitional period) and the ovulatory season (ovulatory period) in eight mares using a cross-over experimental design within periods. Granulosa cells were collected from follicles 15-24 or 25-34 mm and expression of IGF1R, IGF2R, FSHR, LHCGR and PAPPA was determined by qPCR. In addition, 10 mg IGF1 or vehicle were injected into the largest follicle (transitional period) or the second largest follicle (ovulatory period) of a follicular wave before the beginning of diameter deviation between the two largest follicles (mean diameters at injection 19.2 and 20.0 mm during transitional and ovulatory periods respectively). Follicular fluid was collected 24 h after injection for determination of free IGF1, IGFBP, inhibin A and oestradiol levels. Granulosa cells from follicles 25-34 mm, but not follicles 15-24 mm, expressed higher levels of IGF1R (P=0.01), FSHR (P<0.007) and LHCGR (P=0.09) during the ovulatory period than during the transitional period, whereas IGF2R expression was higher in transitional than ovulatory follicles (P=0.06). Follicular IGFBP2 levels were not different (P>0.1) between periods and treatments, whereas IGFBP5 levels were higher (P<0.05) during the ovulatory period. Finally, IGF1 injection before the beginning of deviation induced an approximately twofold increase (P=0.01) in follicular inhibin A levels during each period and did not affect oestradiol (P>0.1). These results suggest that, as during ovulatory waves, equine follicles during transitional waves are responsive to IGF1 before the beginning of deviation and that, therefore, inadequate IGF1 responsiveness before deviation may not underlie the deficient development of dominant follicles during transition.     

Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression

The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function.     

Determination of the pigmenting carotenoids in feeds and premixes for laying hens and broilers (author's transl)

A method for the quantitative determination of the main carotenoids, such as lutein, zeaxanthin, apocarotenoic acid ethyl ester, bixin, canthaxanthin, citranaxanthin and paprika pigments in feeds and premixes for laying hens and broilers is described. The method has, compared to the known analytical procedures, several advantages: 1. Differentiation between lutein and zeaxanthin which have different pigmentation properties. 2. Reduction of distribution errors for added carotenoids by increasing sample weights. 3. Individual determinations of added carotenoids which have different pigmentation activities.     

Determination of carnosine in Black-Bone Silky Fowl (Gallus gallus domesticus Brisson) and common chicken by HPLC

Black-Bone Silky Fowl (Gallus gallus domesticus Brisson) is both consumed as a healthy food and used particularly as a kind of traditional Chinese medicine. Carnosine in the muscles of Black-Bone Silky Fowl was identified by HPLC–MS/PAD. The carnosine content in the meat of Black-Bone Silky Fowl was determined by HPLC and compared with that contained in the meat of White Plymouth Rock, which were bred under the same condition. The results showed that the contents of carnosine in the mixed meat, breast meat and thigh meat of Black-Bone Silky Fowl were all remarkably higher than that in the White Plymouth Rock. These findings indicate that Black-Bone Silky Fowl would be a better chicken breed for carnosine supplement.     

Regulation of ovarian follicular growth by somatotropin and insulin-like growth factors in cattle

Somatotropin (ST), insulin-like growth factor (IGF)-I, and IGF-II affect animal growth and lactation as well as animal reproduction. Understanding the effects of ST and the IGF on reproduction is important because ST and IGF-I undergo dynamic changes prior to the postpartum breeding period. In addition, administration of recombinant bovine somatotropin (rbST) to lactating cows is a common practice that increases blood concentrations of ST and IGF-I during the breeding period. In vivo, administration of rbST caused greater ovarian follicular development. The effects of rbST may represent direct actions of ST because ST receptors are found within granulosa cells as well as oocytes. Alternatively, the actions of ST may be indirectly mediated by increased IGF-I and (or) nutrient partitioning that occurs after rbST. Both IGF-I and IGF-II are synthesized within the ovary. Ovarian IGF are, therefore, a composite of IGF from both endocrine (liver) and autocrine and paracrine (ovary) sources. The IGF stimulate ovarian function by acting synergistically with gonadotropins to promote growth and steroidogenesis of ovarian cells. Actions of IGF-I and -II are restrained by a series of IGF binding proteins (IGFBP) that either originate from the blood or are synthesized locally within the follicle. Degradation and differential synthesis of IGFBP are important mechanisms regulating IGFBP amounts. The relative amounts of IGFBP may ultimately determine ovarian IGF action. Future studies of ST and IGFs should focus on the hormones, receptors, and binding proteins as well as the metabolic requirements for normal ovarian function in dairy cattle.     

Testosterone antagonist (flutamide) blocks ovulation and preovulatory surges of progesterone, luteinizing hormone and oestradiol in laying hens

The preovulatory release of luteinizing hormone (LH) in the domestic hen occurs after the initiation of a preovulatory surge of testosterone. The objective of this study was to determine whether this testosterone surge has functional significance in the endocrine control of ovulation. Groups of laying hens (n = 10-22) were treated with the androgen receptor antagonist, flutamide, at 8 h intervals for 24 h at doses of 0, 31.25, 62.5, 125 and 250 mg. All doses reduced egg laying (P < 0.001), with the highest dose being the most effective. In a second study, laying hens (n = 9) were treated with 250 mg flutamide at 8 h intervals for 24 h with a control group being given placebo (n = 10). Blood samples were taken for hormone measurements at 2 h intervals for 18 h starting 4 h before the onset of darkness. The percentage of hens laying per day did not differ between groups before treatment (control, 88% vs flutamide, 86%). Ovulation was blocked in all hens treated with flutamide within 2 days while the control hens continued to lay at the pretreatment rate (80%). Preovulatory surges of plasma testosterone, progesterone, oestradiol and LH were observed in control hens but with the exception of testosterone, flutamide treatment blocked the progesterone, oestradiol and LH surges. LH concentrations declined progressively with time in the flutamide-treated hens. It is concluded that inhibition of testosterone action blocks egg laying and the preovulatory surges of progesterone, luteinizing hormone and oestradiol demonstrating a key role for the preovulatory release of testosterone in the endocrine control of ovulation in the domestic hen.     

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.     

Changes in the carbohydrate constituents of yam tuber (dioscorea rotundata,poir) during growth

The amounts of sugars, starch, hemicellulose and cellulose in yam tubers were estimated at different stages of maturity and in yam tuber (“EgbodO”) sections. Maltose, sucrose, glucose and fructose were the free sugars identified in the ethanolic extracts of the yam samples. Sucrose formed the bulk of the total sugars. The “bottom” portion of the “Egbodo” yam tuber was richer in total sugars (4.9%) than either the “middle” (1.8%) or the “head” (2.4%) regions. Starch accounted, respectively, for 60, 77 and 78% of total carbohydrates in the “bottom”, “middle” and “head” regions of the “egbodo” yam tuber. A peak value of 84% starch was obtained 6 months after planting the yam setts. Its decrease to 64% at 8 months has been partly attributed to reduced photosynthetic activity through yellowing and subsequent loss of leaves. The sum of cellulose and hemicellulose, regarded as non-available carbohydrates was less than 7% of the total carbohydrates. The linear fraction, amylose, formed about 17.8% of the starch prepared from whole yam tubers of varying maturities, and from yam tuber sections, the remaining being amylopectin. The observed amylose and amylopectin values did not vary significantly over the period of observation. These amylose and amylopectin fractions had iodine affinities of 17 and 0.1%, respectively.     

Dose-response feeding study of short chain chlorinated paraffins (SCCPs) in laying hens: effects on laying performance and tissue distribution, accumulation and elimination kinetics

Technical short chain chlorinated paraffins (C10-C13 with 60% chlorine) were fed to 93 laying hens from 24 to 32 weeks of age in increasing concentrations of up to 100 mg/kg feed. No significant influence on health, relative organ weights or performance (laying intensity, egg weight, feed consumption) was noted. The chlorinated paraffin content of the tissues was linearly related to the concentration of short chain paraffins of the feed. The highest concentrations were found in abdominal fat, egg yolk and fatty tissues. Breast muscle, egg albumen and bile fluid contained minimal or no residues. Less than 1% of the chlorinated paraffins ingested were incorporated into the body (without head, feet, gut and feathers), whereas about 1.5% were eliminated with the egg yolk and 30% were excreted with urine and faeces. A six-week kinetic depuration study revealed a biphasic elimination with half-lifes of 4-40 min (liver, kidneys, legs, fat, blood) for the initial rapid phase, and 15-30 days (blood, fat, liver, yolk, kidneys, legs) for the terminal slow phase.     

Changes in the nitrogen fraction of sainfoin (Onobrychis viciaefolia) during growth

Sainfoin was harvested at four stages of increasing maturity. Samples were analysed for total N, true protein N, non-protein N, nitrate and amino acids. Variations in the concentrations of these nitrogenous fractions throughout the growing period of plant are described.     

产抗菌肽乳酸菌筛选及抗菌肽的分离纯化与特性研究

目的筛选得到产抗菌肽菌株,对抗菌肽进行分离纯化及理化性质研究。方法利用琼脂扩散法筛选产抗菌肽的菌株,运用细胞吸附解吸和阳离子交换层析法纯化抗菌肽,从热稳定性、酸碱稳定性和蛋白酶敏感性3方面研究抗菌肽的理化特性。结果筛选得到7株产抗菌肽的菌株,其中菌株4121具有较广的抑菌谱,命名为鼠李糖乳杆菌4121。对抑菌物质进行分离纯化和理化性质测定,电泳结果显示抗菌肽的分子量约为11 kDa,最终获得的抗菌肽比活力为12379.11 AU/mg,最终纯化倍数为91.99倍。该抗菌肽的抑菌能力在20~80℃及偏酸条件下具有很好的稳定性。同时该抗菌肽对蛋白酶K和α-胰凝乳蛋白酶敏感。结论筛选出的鼠李糖乳杆菌4121所产生的抗菌肽热稳定性及酸稳定性良好,具有广谱抑菌能力,并建立了有效的抗菌肽分离纯化法—细胞吸附解吸-阳离子交换层析法。     

Purification and identification of novel antioxidative peptide released from Black-bone silky fowl (Gallus gallus domesticus Brisson)

Papain-treated Black-bone silky fowl (BSF) muscle hydrolysate was subjected to 6 kDa cutoff membrane ultrafiltration, and the resulting BSF peptides (<6 kDa) were purified by two-step reverse-phase high-performance liquid chromatography. The molecular weight (MW) distribution and amino acid composition were investigated for characterization of the BSF peptides. The results showed that the major amino acids of BSF peptides were Glu, Tyr, Lys, Asp, Leu, Ala, Thr and Pro, and the MW was from 281 to 7,982 Da. BSF peptides exhibited a strong antioxidant capacity. At 10 mg/mL, they displayed more powerful $ {\text{O}}_{2}^{ \cdot - } $ , DPPH^· and ABTS^·+ scavenging activity and reducing power than carnosine. The peptide fraction 8 with more hydrophilicity revealed stronger $ {\text{O}}_{2}^{ \cdot - } $ and ABTS^·+ scavenging activity and reducing power than BSF peptides and carnosine. Besides, a peptide, separated from fraction 8 and showed the strongest antioxidant capacity, was purified and identified by LC-ESI-MS/MS to be Glu-Pro-Asp-Arg-Tyr (678 Da).