Effects of crocin on frozen-thawed sperm apoptosis,protamine expression and membrane lipid oxidation in Yanbian yellow cattle

Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.     

Recovery of Campylobacter from segments of the reproductive tract of broiler breeder hens

Three groups of >60-wk-old broiler breeder hens were assessed for the presence of Campylobacter within segments of the reproductive tract. In the first group, after stunned, the hens were bled, scalded, and defeathered, the reproductive tracts were aseptically excised from 18 hens, six from each of three adjacent floor pens that were feces positivefor Campylobacter. The reproductive tract segments (infundibulum, magnum-isthmus, shell gland, vagina, and cloaca) were pooled by pen. In the second group, 10 individual hens were sampled from the pens; the reproductive tract was divided into the following segments: magnum, isthmus, shell gland, vagina, and cloaca. For the third group, hens were obtained from two commercial farms that had been determined to be feces positive for Campylobacter, and the reproductive tract was divided into five segments, as described for the second group. Segments of the reproductive tract were placed into sterile plastic bags and suspended 1:3 (w/v) in Bolton enrichment broth, and serial dilutions were plated (0.1 ml) onto Campy-Cefex agar. The agar places were incubated at 42 C for 24 hr in a microaerobic atmosphere. In group 1, the pooled reproductive tract segments for hens from pen A were Campylobacter positive for the shell gland, vagina, and cloaca; hens from pen B were positive for the cloaca only; and hens from pen C were positive for the magnum-isthmus and doaca. In the second group, 9 of 10 cloaca samples were Campylobacter positive. Commercial hens in group 3 had campylobacter-positive cloaca samples (12/12), vagina (10/12), shell gland (7/12), isthmus (2/12), and magnum (4/12). Campylobacter colonization of the reproductive tract of the hen could enable vertical transmission of Campylobacter from the hen to the chick.     

Effects of Constant, 9 and 16-h Light Cycles on Sperm Quality, Semen Storage Ability and Motile Sperm Subpopulations Structure of Boar Semen

This study was performed to test the effect that two separate, daily, constant-light regimes of both 9 and 16 h could have on the main parameters of boar-semen quality analysis, as well as on the motile sperm subpopulations structure and the ability of its conservation at 16 degrees C. Results show that both luminous regimes have slight, specific effects on the main parameters of boar-semen quality analysis, as well as on the motile sperm subpopulations structure. Furthermore, the conservation ability at 16 degrees C of boar semen was not significantly different between both photoperiods. When a temporal study was performed, results showed that semen quality and motility parameter changes were stabilized at nearly constant values from the second month of the study to the last month in both luminous regimes, indicating a rapid light-related effect on testicular function. Our results indicate that light regimes oscillating from 9 h daily to 16 h daily are of little importance in the control of boar-semen quality in a farming environment.     

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre‐Implantation Stage

Oocyte has been considered the major contributor for embryo thermo‐tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo‐tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo‐sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post‐insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat‐shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat‐shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.     

Transmission studies of Hendra virus (equine morbilli-virus) in fruit bats, horses and cats

Objective To determine the infectivity and transmissibility of Hendra virus (HeV). Design A disease transmission study using fruit bats, horses and cats. Procedure Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second exper iment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experi ment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. Results Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated devel oped acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. Conclusions Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus t o horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.     

Temperature as a universal resetting cue for mammalian circadian oscillators

Environmental temperature cycles are a universal entraining cue for all circadian systems at the organismal level with the exception of homeothermic vertebrates. We report here that resistance to temperature entrainment is a property of the suprachiasmatic nucleus (SCN) network and is not a cell-autonomous property of mammalian clocks. This differential sensitivity to temperature allows the SCN to drive circadian rhythms in body temperature, which can then act as a universal cue for the entrainment of cell-autonomous oscillators throughout the body. Pharmacological experiments show that network interactions in the SCN are required for temperature resistance and that the heat shock pathway is integral to temperature resetting and temperature compensation in mammalian cells. These results suggest that the evolutionarily ancient temperature resetting response can be used in homeothermic animals to enhance internal circadian synchronization.     

Effects of Intrauterine Devices in Mares: A Histomorphological and Immunohistochemical Evaluation of the Endometrium

Oestrous suppression by intrauterine devices (IUDs) is caused by prolongation of luteal function, but the biological mechanism is unknown. The aim of the study was to investigate mechanisms which could explain the action of IUDs. Thirty mares were age‐matched and either inseminated (AI, n = 15) or fitted with an IUD (IUD, n = 15) and subsequently divided into four groups: AI‐P, pregnant (n = 8); AI‐N, non‐pregnant (n = 7); IUD‐P, prolonged luteal phase (n = 7); and IUD‐N, normal luteal phase (n = 8). The median ages were 5.5 and 7 years in AI‐P and IUD‐P groups and 14 and 11 years in AI‐N and IUD‐N groups, respectively. On Day 15 after ovulation, an endometrial biopsy was obtained to study histomorphological and immunohistochemical expression patterns of uterine proteins (uteroferrin, UF; uterocalin, UC; uteroglobin, UG), oestrogen and progesterone receptors (ER, PR), proliferation marker Ki‐67 and content of inflammatory cells. Expression of UF was higher in IUD mares; the difference between pregnant and IUD‐P mares was significant. Mares exhibiting a prolonged luteal phase (AI‐P, IUD‐P) showed only mild angiosclerosis and lower expression of both ER and PR than mares with a normal luteal phase (AI‐N, IUD‐N). No significant differences were detected in the numbers of inflammatory cells, with the exception of macrophages, which were more numerous in AI‐P than AI‐N mares. Although inflammatory cells were not detected in IUD mares, increased UF levels may indicate chronic inflammation. Young age and normality of the endometrial blood vessels may improve the efficacy of IUDs.     

Horizontal transmission of Salmonella and Campylobacter among caged and cage-free laying hens

In each of five sequential trials, laying hens (56-72 wk of age) were challenged with Salmonella and Campylobacter, and 1 wk postinoculation, the challenged hens (n = 3) were commingled with nonchallenged hens (n = 12) in conventional wire cages, on all-wire slats, or on all-shavings floor housing systems. After 12 days, challenged and nonchallenged hens were euthanatized for sample collection. Ceca were aseptically collected from all hens, and the spleen, liver/gallbladder (LGB), lower (LRT) and upper (URT) reproductive tracts, and ovarian follicles (mature and immature) were collected from only the challenged hens after commingling. Samples were divided equally and cultured separately for Salmonella and Campylobacter. Differences in the horizontal transmission of the challenge Salmonella to nonchallenged hens housed in cages (12%), on slats (15%), and on shavings (14%) were not significantly different (P > 0.05) from the challenged pen-mate hens over the five trials. However, with the inclusion of residual environmental Salmonella, the recovery of Salmonella from nonchallenged hens housed in cages was lowest at 15%, intermediate for hens on slats at 20%, and highest for hens on shavings at 38%. Among challenged hens housed in cages, Salmonella was recovered from only 27% of the cecum and LRT samples. From challenged hens housed on slats, Salmonella was recovered from 38% of the cecum, 12% of the spleen, 19% of the LGB, 44% of the LRT, and 19% of the URT samples. From challenged hens housed on shavings, Salmonella was recovered from 31% of the cecum; 15% of the spleen, LGB, and URT; and 31% of the LRT samples. Horizontal transmission of Campylobacter among nonchallenged pen-mate hens was significantly lower for hens housed in cages at 28% than for hens on shavings at 47%, with hens on slats being intermediate at 36%. For challenged hens housed in cages, Campylobacter was recovered from 27% of the cecum, 13% of the LRT, 7% of the URT, and 17% of the follicle samples. Among the challenged hens housed on slats, Campylobacter was recovered from 44% of the cecum, 6% of the spleen, 19% of the LGB, 12% of the LRT, 6% of the URT, and 14% of the follicle samples. Among challenged hens housed on shavings, Campylobacter was recovered from 46% of the cecum, 8% of the LRT and URT, and 40% of the follicle samples. The overall results of this study indicate that the caged housing system provided the lowest horizontal transmission level of Salmonella and Campylobacter among egg-laying hens.