Rosemary extract improves egg quality by altering gut barrier function,intestinal microbiota and oviductal gene expressions in late-phase laying hens

Background Rosemary extract(RE)has been reported to exert antioxidant property.However,the application of RE in late-phase laying hens on egg quality,intestinal barrier and microbiota,and oviductal function has not been systematically studied.This study was investigated to detect the potential effects of RE on performance,egg quality,serum parameters,intestinal heath,cecal microbiota and metabolism,and oviductal gene expressions in late-phase laying hens.A total of 21065-week-old“Jing Tint 6”laying hens were randomly allocated into five treatments with six replicates and seven birds per replicate and fed basal diet(CON)or basal diet supplemented with chlortetracycline at 50 mg/kg(CTC)or RE at 50 mg/kg(RE50),100 mg/kg(RE100),and 200 mg/kg(RE200).Results Our results showed that RE200 improved(P<0.05)Haugh unit and n-6/n-3 of egg yolk,serum superoxide dismutase(SOD)compared with CON.No significant differences were observed for Haugh unit and n-6/n-3 of egg yolk among CTC,RE50,RE100 and RE200 groups.Compared with CTC and RE50 groups,RE200 increased serum SOD activity on d 28 and 56.Compared with CON,RE supplementation decreased(P<0.05)total cholesterol(TC)level.CTC,RE100 and RE200 decreased(P<0.05)serum interleukin-6(IL-6)content compared with CON.CTC and RE200 increased jejunal m RNA expression of ZO-1 and Occludin compared with CON.The biomarkers of cecal microbiota and metabolite induced by RE 200,including Firmicutes,Eisenbergiella,Paraprevotella,Papillibacter,and butyrate,were closely associated with Haugh unit,n-6/n-3,SOD,IL-6,and TC.PICRUSt2 analysis indicated that RE altered carbohydrate and amino acid metabolism of cecal microbiota and increased butyrate synthesizing enzymes,including 3-oxoacid Co A-transferase and butyrate-acetoacetate Co A-transferase.Moreover,transcriptomic analysis revealed that RE200 improved gene expressions and functional pathways related to immunity and albumen formation in the oviductal magnum.Conclusions Dietary supplementation with 200 mg/kg RE could increase egg quality of late-phase laying hens via modulating intestinal barrier,cecal microbiota and metabolism,and oviductal function.Overall,RE could be used as a promising feed additive to improve egg quality of laying hens at late stage of production.

Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality

Background:In vitro production of bovine embryos is a well-established technology,but the in vitro culture(IVC)system still warrants improvements,especially regarding embryo quality.This study aimed to evaluate the effect of extracellular vesicles(EVs)isolated from oviductal(OF)and uterine fluid(UF)in sequential IVC on the development and quality of bovine embryos.Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum(dFCS)in the presence(BSA-EV and dFCS-EV)or absence of EVs from OF(D1 to D4)and UF(D5 to D8),mimicking in vivo conditions.EVs from oviducts(early luteal phase)and uterine horns(mid-luteal phase)from slaughtered heifers were isolated by size exclusion chromatography.Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents,mitochondrial activity and total cell numbers,as well as survival rate after vitrification.Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormonesensitive lipase(pHSL)proteins were also determined.Additionally,the expression levels of 383 miRNA in OF-and UF-EVs were assessed by qRT-PCR.Results:Blastocyst yield was lower(P<0.05)in BSA treatments compared with dFCS treatments.Survival rates after vitrification/warming were improved in dFCS-EVs(P<0.05).EVs increased(P<0.05)blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls(dFCS and BSA),while lipid content was decreased in dFCSEV(P<0.05)and mitochondrial activity did not change(P>0.05).Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium(PPARGC1B,LDLR,CD36,FASN and PNPLA2,P<0.05).Levels of pHSL were lower in dFCS(P<0.05).Twenty miRNA were differentially expressed between OF-and UF-EVs and only bta-miR-148b was increased in OF-EVs(P<0.05).Conclusions:Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming,total cell number,lipid content,and relative changes in expression of lipid metabolism transcripts and lipase activation.Finally,EVs miRNA contents may contribute to the observed effects.

The sperm-interacting proteome in the bovine isthmus and ampulla during the periovulatory period

Background Spermatozoa interact with oviduct secretions before fertilization in vivo but the molecular players of this dialog and underlying dynamics remain largely unknown.Our objectives were to identify an exhaustive list of sperm-interacting proteins(SIPs)in the bovine oviduct fluid and to evaluate the impact of the oviduct anatomical region(isthmus vs.ampulla)and time relative to ovulation(pre-ovulatory vs.post-ovulatory)on SIPs number and abundance.Methods Pools of oviduct fluid(OF)from the pre-ovulatory ampulla,pre-ovulatory isthmus,post-ovulatory ampulla,and post-ovulatory isthmus in the side of ovulation were collected from the slaughterhouse.Frozen-thawed bull sperm were incubated with OF or phosphate-buffered saline(control)for 60 min at 38.5℃.After protein extraction and digestion,sperm and OF samples were analyzed by nanoLC-MS/MS and label-free protein quantification.Results A quantitative comparison between proteins identified in sperm and OF samples(2333 and 2471 proteins,respectively)allowed for the identification of 245 SIPs.The highest number(187)were found in the pre-ovulatory isthmus,i.e.,time and place of the sperm reservoir.In total,41 SIPs(17%)were differentially abundant between stages in a given region or between regions at a given stage and 76 SIPs(31%)were identified in only one region×stage condition.Functional analysis of SIPs predicted roles in cell response to stress,regulation of cell motility,fertilization,and early embryo development.Conclusion This study provides a comprehensive list of SIPs in the bovine oviduct and evidences dynamic spatiotemporal changes in sperm-oviduct interactions around ovulation time.Moreover,these data provide protein candidates to improve sperm conservation and in vitro fertilization media.

Evaluation of Tubal Patency with Transvaginal Three-dimensional Hysterosalpingo-contrast Sonography

Objective To investigate diagnostic efficacy of transvaginal three-dimensional hysterosalpingo-contrast sonography(3D-Hy Co Sy) in assessing tubal patency with chromolaporoscopy. Methods A total of 157 infertile women underwent 3D-Hy Co Sy to evaluate tubal patency. Among these patients, 39 patients were also examined by chromolaporoscopy. The concordance of the two clinical assessment methods was analyzed by the Kappa coefficient test. Results Among the 306 oviducts examined by 3D-Hy Co Sy, 99(32.4%) were patent, 126(41.2%) partially obstructed, and 81(26.5%) completely obstructed. Diagnostic results with 3D-Hy Co Sy were not statistically different from those obtained in the 39 women(78 oviducts) who also underwent chromolaporoscopy, and the two methods showed a high concordance(κ=0.747, P=0.000). The 3D-Hy Co Sy procedure had a sensitivity of 84.8%(28/33), a specificity of 96.2%(25/26), and positive and negative predictive values of 93.3%(28/30) and 86.2%(25/29) respectively. Conclusion Transvaginal 3D-Hy Co Sy can accurately reveal the spatial path and morphology of the oviduct and is a safe and effective method to evaluate tubal patency.

Structural changes of oviduct of freshwater shrimp, Macrobrachium nipponense (Decapoda,Palaemonidae), during spawning

The structural change of the oviduct of freshwater shrimp (Macrobrachium nipponense) during spawning was ex- amined by electron microscopy. The oviduct wall structural characteristics seem to be influenced significantly by the spawning process. Before the parturition and ovulation, two types of epithelial cells (types I and II) are found in the epithelium. The free surfaces of type I and type II cells have very dense long microvilli. Under the type I and type II cells, are a relatively thick layer of secreting material and a layer of mostly dead cells. After ovulation, two other types of epithelial cells (types III and IV) are found in the oviduct wall epithelium. The free surface of type III cells only has short microvilli scattered on the surface. The thick layer with secreting material and the dead cell layer disappeared at this stage. In some type III cells, the leaking out of cytoplasm from broken cell membrane led to the death of these type III cells. The transformation of all four types of epithelial cells was in the order: IV→I→II→III.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Laparoscopic Embryo Transfer in Pigs:Knowledge for Surgical Procedures

The objective of the study was to describe and improve a technique for laparoscopic embryo transfer into the oviduct and uterine horns of pigs and to evaluate the feasibility and safety of this method. Fourteen female pigs were randomly allocated into groups A and B: three portals were used for group A, and the simulation of embryo(0.9% Na Cl, 0.2 m L) was injected into the tip of uterine horn; three or four portals were used for group B, and the injection set of the oviduct was inserted through the abdominal orifice of uterine tube into the oviduct to inject the simulation of embryo. The repeat laparoscopy was performed on the 21 st day. Three pigs randomly selected from each groups were repeated the same procedure three times, and then were euthanized on the 21 st day after the last surgery and a complete necropsy performed. Laparoscopic embryo transfer was performed successfully in all pigs without major intra-and post-operative complications. The average surgical time which accomplished procedures for groups A and B was 18.6 min(range, 28-14 min) and 37.4 min(range, 53-29 min). Postoperatively, none of pigs appeared to abnormal signs. This study demonstrated that laparoscopic embryo transfer could be easily accomplished by using the special-purpose equipment and increasing reuse time of a female recipient.

Endometrial clear cell carcinoma invading the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor:A case report

BACKGROUND To investigate the clinicopathological features of endometrial clear cell carcinoma that has invaded the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor.CASE SUMMARY A case of endometrial clear cell carcinoma invading the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor was collected and analyzed using pathomorphology and immunohistochemistry.Endometrial clear cell carcinoma cells were distributed in a solid nest,papillary,shoe nail-like,and glandular tube-like distribution.There was infiltrative growth,and tumor cells had clear cytoplasm and obvious nuclear heteromorphism.The cancer tissue was necrotic and mitotic.The cancer tissue invaded the right oviduct.The ipsilateral oviduct also had an adenomatoid tumor.The adenomatoid tumor was arranged in microcapsules lined with flat or cubic cells that were surrounded by smooth muscle tissue.The adenomatoid tumor cells were round in shape.CONCLUSION Clear cell carcinoma of the endometrium can invade the oviduct and occur simultaneously with tubal adenomatoid tumors.Upon pathological diagnosis,one should pay close attention to distinguishing whether an endometrial clear cell carcinoma is invading the oviduct or whether it is accompanied by an adenomatoid tumor of the oviduct.Immunohistochemistry is helpful to differentiate these two disease entities.Endometrial clear cell carcinomas express Napsin-A and P16 and are negative for estrogen receptor and progesterone receptor.The presence of endometrial clear cell carcinoma does not affect the expression of CK and calretinin in adenomatoid tumors.

In vivo and in vitro development of Tibetan antelope(Pantholops hodgsonii)interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau,China,and is now considered an endangered species.As a possible rescue strategy,the development of embryos constructed by interspecies somatic cell nuclear transfer(iSCNT)was examined.Tibetan antelope fibroblast cells were transferred into enucleated bovine,ovine and caprine oocytes.These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals.Less than 0.5%of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage.However,when the cloned antelope-bovine embryos were transferred to caprine oviducts,about 1.6%of the embryos developed to the blastocyst stage.In contrast,only 0.7%of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts.The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine.When the antelope-bovine embryos,constructed from oocytes treated with roscovitine or trichostatin A,were cultured in rabbit oviducts 2.3%and 14.3%developed to blastocysts,respectively.It is concluded that although some success was achieved with the protocols used,interspecies cloning of Tibetan antelope presents difficulties still to be overcome.The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

Changes in oviduct structure in the black tiger shrimp,Penaeus monodon,during ovarian maturation

Objective:To examine the structure of the oviduct of the shrimp Penaeus monodon.Methods:The oviducts of P.monodon with three different major groups of ovarian development(Group(Gr.) 1:Stages I & V;Gr.2:Stages II & III;and Gr.3:Stage IV) were examined by light,transmission electron,and scanning electron microscopies,respectively.Results:The epithelium of the oviduct in Gr.1 was composed of tall simple columnar cells with their basal nuclei located on the basement membrane and its thick collagen fibers.In Gr.2,the oviduct seemed to produce some substances and their epithelial cells became transitional with centrally located nuclei and formed some vacuoles.Obviously,the epithelial cells in Gr.3(at Stage IV) were disorganized,disrupted,and shed accumulated spherical secretory substances including some cellular contents into the lumen.Conclusions:The structural changes of the P.monodon oviduct were related to ovarian maturation stages(Grs.1-3).Prior to spawning,only the oviduct epithelium at ovary Stage IV produced and secreted a number of spherical secretion substances into the lumen.These substances may act as the oviductal lubricants to facilitate the spawning process.

Clinical Observation on 115 Patients of Oviduct Pregnancy Treated by Trichosanthin Injection

Objective: To find an effective treatment of oviduct pregnancy. Methods: One hundred and fifteen patients of oviduct pregnancy were treated by injecting trichosanthin in cervlx. Results: One week after injection, 95.5 % of the treated patients had their HCG turned to negative. Symptoms such as painful abdominal distention, and signs such as pelvic hydrops and vaginal bleeding, were also relieved obviously in most of the cases. Conclusion: Trichosanthin injection may be an effective treatment for oviduct pregnancy, but further improvement in its preparation, and method of medication and dosage are necessary.