Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin

Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (βCD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3-4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane “fluidity” of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM β-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the “fluidity” of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.     

Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.     

Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation

The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 μM MnTE significantly improved membrane integrity while 0.01 μM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 μM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage.     

Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation

The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 μM MnTE significantly improved membrane integrity while 0.01 μM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 μM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage.     

Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin

Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (beta CD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3-4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane "fluidity" of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM beta-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the "fluidity" of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.     

冷冻对山羊精子转染外源DNA和体外制备转基因胚胎的影响

本实验将鲜精和冻精分别与地高锌标记的线形化的pEGFP-N,质粒孵育转染,用原位杂交方法检测转染效率;PCR和Southern Blotting检测精子与外源DNA的整合效率;与成熟卵母细胞体外受精,PCR检测阳性胚胎比率,用透射电镜技术、碘化丙锭和羟化荧光素双探针技术和单细胞电泳（Single Cell Gel Electrophoresis,SCGE）技术,观察精子冷冻前后的超微结构、精子质膜完整性和精子核DNA损伤的变化,研究冷冻对山羊（Caprahircus）精子转染内化外源DNA和体外制备转基因胚胎的影响及机理。结果表明,冻精显著提高了转染外源DNA的效率（81．60％&#177;16．59％VS32．95％&#177;2．93％,t=4．873,P=0．003;41．80％&#177;6．26％vs27．89％&#177;8．64％,t=2．634,P=0．039）。PCR和Southern Blotting检测表明外源DNA已经整合到精子基因组上。用冻精与成熟卵母细胞体外受精,体外受精穿透率和卵裂率显著低于鲜精组（24．19％&#177;3．15％vs58．86％&#177;3．73％,t=7．131,P〈0．001;11．83％&#177;2．37％vs29．71&#177;3．47％,t=4．302,P〈0．001）,但体外生产的胚胎PCR阳性率比鲜精组显著提高（45．45％&#177;10．87％VS24．44％&#177;6．06％,t=1．750,P=0．013）。超微结构观察和双荧光探针检测都发现冷冻-解冻精子质膜完整性降低（8．34％&#177;4．21％VS65．67％&#177;6．46％,t=12．492,P〈0．001）,SCGE显示冷冻极显著增加了精子彗尾长度和彗星细胞比例（42．67μm&#177;4．56μmvs21．14／Lm&#177;2．36μm,t=5．644,P=0．005;60．00％&#177;4．00％vs17．37％&#177;2．57％;t=15．787,P〈0．001）。冷冻-解冻可以提高山羊精子转染外源DNA的效率,冷冻破坏精子质膜完整性,解除质膜的阻碍作用,是提高外源DNA转染效率的一个主要原因[动物学报54（6）：1089-1097,2008]。     

Arbutin's suppression of cryodamage in goat sperm and its mechanism of cryoprotection

Arbutin (4-hydroxyphenyl-glucopyranoside) is a glycosylated hydroquinone present in high concentrations in the leaves of several plants capable of surviving prolonged, extreme dehydration. Two experiments were conducted to determine the effects of arbutin on cryopreservation of goat sperm. In Experiment 1, goat sperm were frozen in extenders with various ratios of Tris-citric acid-glucose (TCG) and arbutin; concentrations of the latter were 0.0 (only TCG), 0.1, 0.2, 0.3, and 0.4 M (only arbutin)]. All extenders had 20% (v/v) egg yolk (EY) and 4% (v/v) glycerol (osmolality = 370 mOsm, pH = 7.0). Sperm motility and acrosome integrity were assessed using CASA, and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA), respectively. Percentages of motile and progressively motile sperm improved with the addition of arbutin; results were optimal (89.0 and 70.0%, respectively; P < 0.05), with 0.4 M arbutin. Furthermore, arbutin improved (P < 0.05) post-thaw recovery rates for both motility and progressive motility. After incubation for 3 h, motility of frozen-thawed washed sperm improved (70%, P < 0.05) with arbutin in the extender. The percentage of sperm with an intact acrosome peaked (77.2%, P < 0.05) with 0.4 M arbutin in the extender. In Experiment 2, the percentage of cells with merocyanine 540/Yo-Pro staining was higher in sperm treated with arbutin than with TCG (P < 0.05), with the best result (58.0%) with 0.4 M arbutin; therefore, arbutin increased membrane fluidity. In conclusion, substitution of a TCG-EY diluent composition with arbutin improved freezability of goat sperm (apparently due to increased membrane fluidity). Furthermore removal of arbutin by centrifugation after freezing and thawing increased sperm longevity.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me₂SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV⁻ PI⁻), early apoptotic (AV⁺ PI⁻), dead non-apoptotic (AV⁻ PI⁺) and necrotic (AV⁺ PI⁺). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.     

FSH induced stimulation of catalase activity in goat granulosa cells in vitro

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3 mm), medium (3–6 mm) or large (>6 mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen

The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P < 0.001), and significant changes in sperm chromatin nuclear decondensation (P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation (P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.     

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26 306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.     

The influence of washing Spanish ibex (Capra pyrenaica) sperm on the effects of cryopreservation in dependency of the photoperiod

Extenders containing low concentrations of egg yolk are recommended for cryopreserving ibex spermatozoa. However, the phylogenetic relationship of the Spanish ibex (Capra pyrenaica) with domestic goats suggests that phospholipases in the seminal plasma may have a negative effect on the response to freezing-thawing when egg yolk-based diluents are employed. The aim of the current work was to determine how seminal plasma removal from Spanish ibex semen, collected by electroejaculation over a period of 1 yr, affects its response to freezing-thawing. Semen was collected from six adult ibexes maintained in captivity. The negative effects of freezing-thawing on the quality of sperm motility and on the integrity of the acrosome and plasma membrane were more serious in the nonwashed semen samples than in those from which the seminal plasma had been removed (P < 0.01, P < 0.05, and P < 0.05 respectively). The beneficial effect of removing the seminal plasma was particularly noticeable during the time of decreasing photoperiod. This suggests that ibex semen shows increased phospholipase activity during the rutting season.     

Biological changes in suspension cultures of Taxus cuspidata induced by dimethyl sulphoxide and ethanol

The biological changes in suspension cultures of Taxus cuspidata caused by dimethyl sulphoxide (DMSO) and ethanol, two commonly used solvents for water-insoluble elicitors, were investigated. The activities of peroxidase (POD) and superoxide dismutase (SOD) changed remarkably after the addition of small amount (0.4% (v/v)) of DMSO compared to those of the control culture at 4 h, however, they were less affected by small amount (0.4% (v/v)) of ethanol within 20 h. The biomass, cell viability, contents of intra/extracellular proteins did not change obviously when the amounts of DMSO and ethanol were below 1% (v/v) and 0.4% (v/v), respectively, but they varied significantly when the contents of DMSO and ethanol were 4% (v/v) and 1% (v/v), respectively. Obvious DNA fragmentation occurred in the case of ethanol at 2% (v/v), while no DNA fragments were observed in the case of DMSO at the same concentration level. It is inferred that DMSO below 1% (v/v) is a better solvent for investigating the effects of water-insoluble elicitors at a long-term contact, while ethanol less than 0.4% (v/v) is more suitable for a short-term contact.     

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P = 0.493). However, when thawed semen was subsequently incubated at 37 °C for periods of between 2 and 48 h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ² = 158.6; DF = 3; P < 0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48 h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.     

Chemical variations of the essential oils in flower heads of Chrysanthemum indicum L. from China

The volatile compositions of hydrodistilled essential oils in the flower heads of Chrysanthemum indicum L. from eight populations in China were analyzed by GC/MS. A total of 169 compounds representing 88.79-99.53% of the oils were identified, and some remarkable differences were found in the constituent percentages of the eight populations. The predominant components of the essential oils were 1,8-cineole (0.62-7.34%), (+)-(1R,4R)-camphor (0.17-27.56%), caryophyllene oxide (0.54-5.8%), β-phellandrene (0.72-1.87%), (-)-(1S,2R,4S)-borneol acetate (0.33-8.46%), 2-methyl-6-(p-tolyl)hept-2-ene (0.3-8.6%), 4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-yl acetate (0.17-26.48%), and hexadecanoic acid (0.72-15.97%). The chemotaxonomic value of the essential-oil compositions was discussed according to the results of cluster analysis (CA) and principal-component analysis (PCA). The eight populations were divided into five groups as different chemotypes (Groups A-E), and the scores together with the loadings revealed clearly different chemical properties of each population. In conclusion, GC/MS in combination with chemometric techniques provided a flexible and reliable method for characterizing the essential oils of different populations of C. indicum L.     

Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus

Role of melatonin in regulation of immunity and reproduction has never been studied in detail in goats. The aim of the present study was to explore hormonal regulation of immunity in goats with special reference to melatonin. Plasma of male and female goats (n = 18 per sex per season) was processed for hormonal (estrogen, testostrone, and melatonin) and cytokine (interleukin [IL-2], IL-6, and tumor necrosis factor α) measurements during three seasons, i.e., summer, monsoon, and winter. To assess cell-mediated immune response, percent stimulation ratio of thymocytes was recorded during three seasons. To support and establish the modulation by hormones, Western blot analysis for expressions of melatonin receptors (MT1, MT2), androgen receptor, and estrogen receptor α and estimations of marker enzymes, arylalkylamine N-acetyltransferase for melatonin and 3β-hydroxysteroid dehydrogenase activities for steroidogenesis were performed in thymus. All the hormones and cytokines were estimated by commercial kits. Biochemical, immunologic, and Western blot analyses were done by standardized protocols. We noted a significant increase in estrogen and testosterone levels (P < 0.05) in circulation during monsoon along with melatonin (P < 0.05) presenting a parallel relationship. Expressions of melatonin receptors (MT1 and MT2) in thymus of both the sexes were significantly high (P < 0.01) during winter. Estrogen receptor α expression in female thymus was significantly high during monsoon (P < 0.05). However, androgen receptor showed almost static expression pattern in male thymus during three seasons. Further, both arylalkylamineN-acetyltransferase and 3β-hydroxysteroid dehydrogenase enzyme activities were significantly high (P < 0.05; P < 0.01, respectively) during monsoon. These results suggest that there may be a functional parallelism between gonadal steroids and melatonin as melatonin is progonadotrophic in goats. Cell-mediated immune parameters (percent stimulation ratio of thymocytes) and circulatory levels of cytokines (IL-2, IL-6, and tumor necrosis factor α) were significantly high (P < 0.01) during monsoon. In vitro supplementation of gonadal steroids to T-cell culture suppressed immunity but cosupplementation with melatonin restored it. Further, we may also suggest that reproductive and immune seasonality are maintained by variations in circulatory hormones and local synthesis of melatonin and gonadal steroids. These functional interactions between melatonin and gonadal steroid might be of great importance in regulating the goat immunity by developing some hormonal microcircuit (gonadal steroid and melatonin) in lymphatic organs.     

Studies on the role of goat heart galectin-1 as a tool for detecting post-malignant changes in glycosylation pattern

Galectins are mammalian lectins established to play a crucial role in the progression of various cancer types by the virtue of their differential expression in normal and cancerous cells. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its potential role in the detection of post-malignant changes in glycosylation pattern. When exposed to superoxide radicals generated from a pyrogallol auto-oxidation system, GHG-1 treated erythrocyte suspension released higher amount of oxyhemoglobin than the unagglutinated erythrocytes. The extent of erythrocyte hemolysis was found to be directly proportional to concentrations of hypochlorous acid. GHG-1 was used to detect the change in the β-galactoside expression pattern in erythrocyte membrane from human donors suffering from prostate and breast cancer. No significant change was observed in the hemolysis of lectin agglutinated erythrocytes collected from pre-operated breast cancer patients, whereas significant increase was observed in normal healthy control and post-operated samples. Findings of this study proclaim GHG-1 as an important tool for the detection of post-malignant changes in glycosylation pattern.Abbreviations: Gal-1, galectin-1; GHG-1, goat heart galectin-1; HOCl, hypochlorous acid; OxyHb, oxyhemoglobin     

Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (相似文献