RESUMEN
A monoclonal antibody (4C7/2E4) that binds to an antigen located on the midpiece of turkey spermatozoa has been produced and characterized. The antibody was also reactive to antigens on the midpiece of chicken, Japanese quail, and bobwhite quail spermatozoa but was unreactive to spermatozoa from guinea fowl, human, horse, boar, and bull. Immunogold transmission electron microscopy of turkey spermatozoa showed that the antibody was associated with the outer mitochondrial membrane. Titratable antibody binding was observed for extracts of turkey spermatozoa and isolated mitochondria. The antibody did not recognize proteins separated by SDS-PAGE, but on Western blots of proteins separated by native gradient-PAGE, the antibody recognized a broad band greater than 660 kDa. No effects on fertilizing capacity, embryonic mortality, or hatchability of eggs were observed when the antibody was added to turkey semen prior to insemination. When semen stored for 0, 3, 6, 24, and 48 h was immunolabeled with 4C7/2E4, the percentage of spermatozoa with unlabeled midpieces was significantly increased by 6 h of storage. The number of free mitochondria and damaged midpieces also significantly increased with storage.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mitocondrias/inmunología , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Pavos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Antígenos/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Fertilización , Hibridomas/inmunología , Masculino , Microscopía Inmunoelectrónica , Mitocondrias/ultraestructura , Semen/fisiología , Preservación de Semen/veterinaria , Espermatozoides/inmunologíaRESUMEN
In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI BLAST searches within the GenBank database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.
Asunto(s)
Células Epiteliales/metabolismo , Serina Endopeptidasas/metabolismo , Pavos/metabolismo , Conducto Deferente/citología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Epiteliales/citología , Biblioteca de Genes , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semen/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Pavos/anatomía & histología , Conducto Deferente/metabolismoRESUMEN
Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.
Asunto(s)
Células Epiteliales/enzimología , Semen/enzimología , Serina Endopeptidasas/química , Pavos/metabolismo , Conducto Deferente/enzimología , Animales , Anticuerpos Monoclonales , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Masculino , Ratones/inmunología , Peso Molecular , Semen/citología , Serina Endopeptidasas/aislamiento & purificación , Fijación del Tejido , Conducto Deferente/citologíaRESUMEN
PURPOSE: Many components of seminal plasma play a role in sperm motility by serving as energy sources. Human seminal plasma contains over 30 proteins, including forward motility proteins, antifertility proteins, and coagulation/liquefaction proteins. This study was designed to determine any correlation between motility or fertilization rates and concentrations of fructose, lactic acid, citric acid, carnitine, and protein in human seminal plasma. METHODS: Fertilization rates were determined by in vitro methods. Fructose, lactic acid, citric acid, and carnitine concentrations were ascertained using high performance liquid chromatography. Protein concentration was determined by Bradford assay. RESULTS: Protein concentrations were significantly different as a function of sperm motility levels. Other constituents of human seminal plasma showed an overall correlation, though not significant. No constituent exhibited significant differences as a function of fertility levels. CONCLUSIONS: Protein concentration was significantly lower for samples with high motility. No significant differences between fertility levels and constituents measured were found.
Asunto(s)
Fertilización In Vitro , Semen/química , Motilidad Espermática/fisiología , Adulto , Carnitina/análisis , Ácido Cítrico/análisis , Femenino , Fructosa/análisis , Humanos , Ácido Láctico/análisis , Masculino , Embarazo , Proteínas/análisis , Semen/fisiología , Estadísticas no ParamétricasRESUMEN
Spermatogenesis is a complicated process dependent upon several factors. Formation of a testis requires the interaction of gene-products and hormones (androgens) on pluripotent tissue. In birds, the female is the heterogametic (ZW) sex, but W chromosomal genes do not influence gonadal development in a way similar to the SRY gene on the mammalian Y chromosome. However, autosomal genes such as SRY-like HMG box gene 9 (SOX9) may influence gonadal development. Hormones affect development; male gonads subjected to estrogen form an ovotestis, whereas ovaries exposed to aromatase inhibitors form an atypical testis. Sertoli cell numbers are set early in spermiogenesis, possibly under the influence of follicle-stimulating hormone and thyroid hormone, and this may determine the number of gonial cells that can be supported. Sertoli cells make a number of substances that affect testicular development and function, particularly anti-Müllerian hormone, which inhibits female oviduct formation from the Müllerian anlage, inhibits aromatase activity to stop estrogen production, and possibly stimulates androgen production by Leydig cells. Undifferentiated primordial germ cells (PGC) migrate to the testis and are converted to spermatogonia by factors from gonadal ridge tissue and androgens. The PGC of males in the ovary form oocytes of Z genotype, whereas the female PGC in males form mostly Z sperm (with a few of W genotype). Transmission electron microscopy micrographs of turkey testis are presented, and control of spermatogenesis by hormones and cytokines is discussed. This discussion includes follicle-stimulating hormone, luteinizing hormone, inhibin, activin, follistatin, tumor necrosis factor-alpha, growth factors such as transforming growth factor-beta, interleukins, and interferon. Although information concerning paracrine and autocrine regulation of the avian testis by these substances is sparse, much can be learned from mammalian studies, in which putative roles of each of these substances have been established. How Sertoli cells cause directed apoptosis of spermatogonia using the Fas-ligand, Fas-receptor pathway is reviewed, as well as ways to circumvent this process. A possible role for ubiquitin concerning prevention of heat-induced damage to the testis is presented.
Asunto(s)
Ovario/anatomía & histología , Ovario/fisiología , Aves de Corral , Espermatogénesis , Testículo/anatomía & histología , Testículo/fisiología , Animales , Pollos , Femenino , Masculino , Espermatozoides/fisiología , Struthioniformes , Pavos , Cromosoma YRESUMEN
A monoclonal antibody (mAb) to epitopes on mitochondria from turkey spermatozoa cross-reacted with Japanese quail spermatozoal mitochondria. However, the pattern of binding was different from that observed for turkey sperm. The ultrastructure of quail spermatozoa was examined to determine the reason for this difference in antibody binding pattern. Light microscopy, as well as scanning (SEM) and transmission (TEM) electron microscopy, were used to study the morphology of spermatozoa from Japanese quail. Japanese quail had a sauropsid type of sperm cell, which is typical of nonpasserine birds. The spermatozoa were vermiform in shape, with a maximum width of 0.6 microm and an overall length between 230 and 250 microm. An acrosome (3.7 to 4.5 microm), nucleus (20.8 to 23.8 microm), midpiece (160 to 170 microm), and tail (40 to 60 microm) were observed. The TEM showed an acrosomal cap surrounding a perforatorium that inserted into the nucleus at the posterior end. Only a distal centriole was observed, which gave rise to a central axoneme with a 9+2 microtubular structure. The axoneme was encased by a spiraled mitochondrial sheath in the midpiece region (64 to 74% of the overall length of the sperm), and mitochondria numbers were estimated to be greater than 1,400 per sperm. In contrast, turkey sperm contain short midpieces with only 20 to 30 mitochondria per sperm. Differences in binding patterns of the mAb to turkey mitochondria between quail and turkey sperm were due to the presence of mitochondria on the exceptionally long midpieces of quail sperm.
Asunto(s)
Coturnix/anatomía & histología , Espermatozoides/ultraestructura , Pavos/anatomía & histología , Acrosoma/ultraestructura , Animales , Anticuerpos Monoclonales , Núcleo Celular/ultraestructura , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Inmunohistoquímica , Masculino , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Mitocondrias/ultraestructura , Cola del Espermatozoide/ultraestructuraRESUMEN
A monoclonal antibody (mAb) to epitopes on mitochondria from turkey spermatozoa cross-reacted with Japanese quail spermatozoal mitochondria. However, the pattern of binding was different from that observed for turkey sperm. The ultrastructure of quail spermatozoa was examined to determine the reason for this difference in antibody binding pattern. Light microscopy, as well as scanning (SEM) and transmission (TEM) electron microscopy, were used to study the morphology of spermatozoa from Japanese quail. Japanese quail had a sauropsid type of sperm cell, which is typical of nonpasserine birds. The spermatozoa were vermiform in shape, with a maximum width of 0.6 microm and an overall length between 230 and 250 microm. An acrosome (3.7 to 4.5 microm), nucleus (20.8 to 23.8 microm), midpiece (160 to 170 micro/m), and tail (40 to 60 microm) were observed. The TEM showed an acrosomal cap surrounding a perforatorium that inserted into the nucleus at the posterior end. Only a distal centriole was observed, which gave rise to a central axoneme with a 9+2 microtubular structure. The axoneme was encased by a spiraled mitochondrial sheath in the midpiece region (64 to 74% of the overall length of the sperm), and mitochondria numbers were estimated to be greater than 1,400 per sperm. In contrast, turkey sperm contain short midpieces with only 20 to 30 mitochondria per sperm. Differences in binding patterns of the mAb to turkey mitochondria between quail and turkey sperm were due to the presence of mitochondria on the exceptionally long midpieces of quail sperm.
Asunto(s)
Coturnix/anatomía & histología , Espermatozoides/ultraestructura , Acrosoma/ultraestructura , Animales , Anticuerpos Monoclonales , Núcleo Celular/ultraestructura , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Inmunohistoquímica , Masculino , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Mitocondrias/ultraestructura , Cola del Espermatozoide/ultraestructura , Pavos/anatomía & histologíaRESUMEN
A case is described wherein a turkey male (tom) possessed two vent areas with duplicated cloacae served by one large intestine. Both cloacae were functional in that feces were excreted and semen could be collected from each. The left vas deferens and ureter emptied into the left cloaca, and the right vas deferens and ureter emptied into the right cloaca. This allowed semen from each testis/duct to be collected separately from the corresponding cloaca. Thus, a unique opportunity was presented to collect semen separately from the left and right testis/duct system for semen analysis and fertility determination. Sperm concentration and percentage of dead sperm were not significantly different when semen from left vs. right reproductive tract were compared. The concentrations of spermiophages in semen from both reproductive tracts fell into the range reported for normal semen (0-8 x 10(5)/ml); however, semen from the right side had consistently higher spermiophage concentrations than that from the left side. On the basis of observations from one male made possible by an anatomic anomaly, it appears that the fecundities of the left and right testis/duct systems of the turkey male do not significantly differ and that recruitment of spermiophages into one tract (because of immunologic challenge, etc.) does not necessarily mean that the opposite testis/ducts will respond similarly.
Asunto(s)
Cloaca/anomalías , Semen , Pavos/anomalías , Animales , Masculino , Testículo/citología , Conducto Deferente/citologíaRESUMEN
The mobility of pooled turkey sperm following various storage regimens was assessed by objectively measuring the ability of sperm to penetrate a 2% Accudenz [5-(N-2,3-dihydroxypropylacetamido)-2,4,6-tri-iodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide] solution at 41 C. When semen was diluted with Beltsville poultry semen extender and stored at 5 C with agitation at 150 rpm, sperm mobility declined as the storage interval increased (P < or = 0.05), with mobility scores (mean +/- SEM) of 0.440+/-0.029, 0.374+/-0.031, 0.282+/-0.011, 0.202+/-0.019, and 0.130+/-0.019 for 0-, 3-, 6-, 24-, and 48-h storage, respectively. For a 10-wk fertility trial using the same storage method, sperm mobility and fertilizing capacity of semen were significantly reduced following 24-h storage compared with values for unstored semen. The sperm mobility scores were 0.404+/-0.051 and 0.101+/-0.046 for unstored and 24-h stored semen, respectively, whereas the percentage of fertilized eggs was 95.9+/-5.1 for unstored semen and 48.0+/-5.1 for 24-h stored semen. When caffeine or pentoxifylline was added to semen at 2.5, 5, or 10 mM, no significant effect on sperm mobility was seen, regardless of whether these compounds were added to unstored semen, were present during 6-h storage, or were added following the 6-h storage interval. These studies demonstrate that sperm mobility and fertilizing capacity of pooled turkey semen declines with storage, and that addition of caffeine or pentoxifylline either during or after storage does not affect sperm mobility.
Asunto(s)
Preservación de Semen/veterinaria , Motilidad Espermática/efectos de los fármacos , Pavos , Xantinas/farmacología , Animales , Cafeína/farmacología , Fertilidad , Masculino , Pentoxifilina/farmacología , Factores de TiempoRESUMEN
Turkey peritoneal exudate cells (PEC) and spermiophages (SMO) were assayed for characteristics of macrophages. The PEC elicited by i.p. injection of 3% Sephadex and SMO isolated from semen using Percoll were cultured in Dulbecco's Modified Eagle Medium supplemented with 20% bovine calf serum (DMEM-20) for 24 h at 41 C in 5% CO2 to provide adherent cells for assays. Most PEC and SMO showed esterase activity (99.3 +/- 0.6 and 98.8 +/- 0.9%, respectively), and exhibited nonspecific phagocytosis of carbon (89.5 +/- 3.6 and 95.3 +/- 0.6%, respectively), zymosan (26.5 +/- 7.6 and 24.3 +/- 2.5%, respectively), bacteria (11.3 +/- 0.8 and 9.3 +/- 0.3%, respectively), and opsonized and nonopsonized SRBC. Maximum uptake of SRBC was seen by 2 h for PEC but not until 4 h for SMO. At time of maximum uptake, SRBC were noted in 35 to 40% of PEC but only in 15 to 20% of SMO. Turkey IgG-FITC bound to both PEC and SMO, but goat anti-turkey IgG-FITC bound only to SMO. Increased nitrite was found in turkey semen after 24 h storage, with highest levels in samples in which SMO were added. Nitrite production was demonstrated using adhered PEC, but SMO could not be tested due to low cell numbers. This research clearly identifies SMO as having macrophage-like activities. Accordingly, these cells may possess the ability to process and present antigen via histocompatibility receptors. Such activity could lead to immune directed responses, including antibody production or activation of cytotoxic T-lymphocytes.
Asunto(s)
Macrófagos/fisiología , Semen/citología , Pavos , Animales , Eritrocitos/inmunología , Esterasas/metabolismo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Inmunoglobulina G/sangre , Macrófagos Peritoneales/fisiología , Masculino , Nitritos/metabolismo , Fagocitosis , Receptores Fc/fisiología , OvinosRESUMEN
The effects of perfluorochemical (PFC) oxygen carriers in turkey semen diluents on fertility and hatchability was measured for a 10-wk period. Semen was diluted (1:1) with Beltsville Poultry Semen Extender (BPSE) or BPSE:FC-75, an emulsified mixture of BPSE and perfluorobutyltetrahydrofuran (technical grade) (FC-75) and stored for 24 h at 5 C with agitation (150 rpm) and aeration with either air, nitrogen (100%), or oxygen (100%). Sperm concentration and percentage of dead sperm were determined prior to and after storage. Sperm concentration (8.35 to 9.21 x 10(9) per milliliter) was not significantly affected by the type of diluent or aeration gas, and only stored semen diluted with BPSE: FC-75 and aerated with nitrogen had increased percentage of dead sperm. Diluent type did not affect the percentage of fertilized eggs; however, fertility and hatchability for both diluent treatments with nitrogen aeration was significantly lower (P < or = 0.05) than for the semen treatments with air or oxygen aeration. Hatchability for semen diluted with BPSE:FC-75 and aerated with oxygen (63.7%) was significantly higher (P < or = 0.05) than that for BPSE-diluted semen aerated with oxygen (43.2%). Although use of oxygen carrying fluorocarbon emulsified with BPSE did not further improve fertility when the semen was stored for 24 h while oxygenated and mechanically agitated, a beneficial effect was noted for hatchability. The fact that nitrogen severely depressed fertility confirms that the beneficial effects of agitation are due to oxygenation of the spermatozoa. Therefore, further studies of oxygen carriers in semen are warranted.
Asunto(s)
Fertilización , Polímeros de Fluorocarbono , Nitrógeno/administración & dosificación , Oxígeno/administración & dosificación , Preservación de Semen , Espermatozoides/fisiología , Pavos , Aire , Animales , Embrión de Pollo , Emulsiones , Femenino , Fluorocarburos , Inseminación Artificial/veterinaria , MasculinoRESUMEN
The semen of turkeys with numerous spermiophages was used for isolating spermiophages by density gradient centrifugation. Isolated spermiophages were suspended in Beltsville Poultry Semen Extender (BPSE) and added to semen with low spermiophage numbers to give approximate spermiophage concentrations of: 2 x 10(5)/mL (medium) and 10(6) (high). Semen with no added spermiophages was the control. Samples were diluted to 1:1 with BPSE, and for each spermiophage level (treatment), semen aliquots were either immediately inseminated or stored 6 h at 4 C with agitation (150 rpm) before insemination. Hens were inseminated weekly, and fertility, embryonic mortality, and hatchability of eggs were determined for a 10-wk period. The experiment was performed twice. In Trial 1, there were no differences in fertility between treatments except that fertility for control stored semen was lower (P < or = 0.05) than that for fresh semen (89.27 vs 95.97, respectively; SEM = 2.2). Neither hatchability nor embryonic mortality was affected by spermiophage level in Trial 1. Spermiophages did not affect fertility in Trial 2; however, hatchability for unstored treatments with added spermiophages was significantly lower than for the control. For stored semen, hatchability was significantly (P < or = 0.05) greater for treatments with added spermiophages than for the control. Differences in embryonic mortality in Trial 2 did not relate to adding spermiophages to the semen. No clearly defined detrimental effect of seminal spermiophages was shown in the present experiments.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Fertilidad/fisiología , Muerte Fetal/fisiopatología , Macrófagos/fisiología , Semen/fisiología , Pavos/embriología , Pavos/fisiología , Animales , Centrifugación por Gradiente de Densidad/veterinaria , Macrófagos/citología , Masculino , Semen/citología , Motilidad Espermática/fisiología , Espermatozoides/citología , Factores de TiempoRESUMEN
Spermiophages were isolated from turkey semen using Percoll gradient centrifugation, cultured in Roswell Park Memorial Institute 1640 medium at room temperature, and characterized by transmission electron microscopy. After 1 to 2 h, the cells enlarged and developed numerous motile mitochondria. Over time, the mitochondria appeared to increase in number and were released into the extracellular medium. Few mitochondria were observed in spermiophages from fresh semen. However, there was an apparent increase in the number and size of mitochondria after Percoll isolation, which was more pronounced in cultured spermiophages. Over a period of 3 h in culture, many spermiophages became engorged with mitochondria, which subsequently appeared to be released as blebs pinched off from the surface. The release of mitochondria resulted in spermiophages with large, empty vacuoles, although their remaining cytoplasm was engorged with mitochondria. Many free mitochondria were present in the medium. The results of the current research suggest that isolating and culturing turkey spermiophages elicit mitochondrial biogenesis, which proceeds unabated until the cells are engorged with and release numerous mitochondria. This may be due to conditions under which the spermiophages were cultured or to nonhistocompatibility of these cells in pooled semen.
Asunto(s)
Macrófagos/ultraestructura , Semen/citología , Pavos , Animales , Masculino , Microscopía Electrónica , Mitocondrias/ultraestructuraAsunto(s)
Planes de Asistencia Médica para Empleados/organización & administración , Sistemas de Información Administrativa/tendencias , Sistemas de Identificación de Pacientes/tendencias , Difusión de Innovaciones , Planes de Asistencia Médica para Empleados/tendencias , Credito y Cobranza a Pacientes/tendencias , Tecnología/tendencias , Estados UnidosRESUMEN
In a preliminary study, it was found that aeration of turkey semen diluted 1:2 with a phosphate buffer diluent (PD) and stored for 24 h at 12 to 13 C improved the fertilizing capacity relative to that obtained from PD-diluted semen that was not aerated (63.8 and 14.0%, respectively). Emulsifying PD with an equal volume of the oxygen carrier perfluorobutyltetrahydrofurane (FC-80) and using the resulting emulsion as a diluent further increased the fertility (86.6%), but the best fertility was from unstored, undiluted semen (98.0%). The efficacy of using emulsions of aqueous diluents with FC-80 for 24-h cold storage of turkey semen was studied in detail. When semen diluted with PD:FC-80 emulsions received varying levels of O2 for aeration, the fertilizing capacity of semen aerated with 60 or 100% O2 (86.1 and 89.6%, respectively) was significantly greater (P < or = .05) than that obtained using 20% oxygen (78.5%). The FC-80 emulsions that were made using commercially available diluents in place of the PD were tested with 100% O2 aeration during storage. The percentage fertility obtained from emulsions made with Beltsville Poultry Semen Extender or Lake's 7.1 "mm" Diluent was significantly greater (P < or = .05) than that for PD or Minnesota Turkey Growers Association Extender (90.5 and 90.2% vs 77.6 and 72.6%, respectively). This research demonstrates that perfluorochemicals may be useful for cold storage of turkey semen when emulsified with optimum diluents and aerated with > or = 60% O2.
Asunto(s)
Criopreservación , Fertilidad/efectos de los fármacos , Fluorocarburos , Furanos , Oxígeno/farmacología , Preservación de Semen/métodos , Pavos , Animales , Portadores de Fármacos , Femenino , Masculino , Proyectos Piloto , Factores de TiempoRESUMEN
Employers that will prosper under a revamped health care system will be those that plan and lobby for redesign of medical and flexible benefits and equip themselves with advanced technology to manage reform changes.
Asunto(s)
Planes de Asistencia Médica para Empleados/organización & administración , Sistemas de Información Administrativa/tendencias , Reforma de la Atención de Salud/tendencias , Estados UnidosRESUMEN
A procedure was developed to rapidly isolate functional, intact mitochondria from turkey spermatozoa. Semen was collected from turkeys, pooled, and centrifuged to remove spermiophages and other cells. The sperm cells were then mechanically disrupted with a Dounce homogenizer, sonicated, and centrifuged using a discontinuous Percoll gradient. Electron microscopy revealed morphologically intact mitochondria. The isolated mitochondria exhibited cytochrome oxidase activity, oxygen consumption, and were stained by rhodamine 123, a fluorescent stain specific for functional mitochondria in eukaryotic cells. Mitochondrial DNA (mtDNA) was isolated and purified, and the genome was determined to be 16.457 +/- 0.07 kbp. Restriction fragment patterns were identified using the endonucleases EcoR1, HindIII, and BamH1. Mitochondrial DNA was also purified from turkey liver and testis, and no differences in the restriction enzyme patterns were found between somatic and germ cell mtDNA. It is concluded that mitochondria can be isolated from spermatozoa for metabolic or genetic study.
Asunto(s)
Fraccionamiento Celular/métodos , Mitocondrias , Espermatozoides/ultraestructura , Animales , Enzimas de Restricción del ADN , ADN Mitocondrial/aislamiento & purificación , Masculino , Mitocondrias/ultraestructura , PavosRESUMEN
1. Continuous 5 hr infusion of low levels of corticosterone, epinephrine, isoproterenol or phenylephrine plus a high dose bolus injection at 3 hr had different effects on the plasma glucose levels of chickens and turkeys. 2. Corticosterone had no effect on plasma glucose in turkeys, but increased glucose after the bolus and at 270 and 300 min for chickens. 3. Epinephrine increased plasma glucose in turkeys, but only caused a transient elevation after the bolus in chickens. 4. Isoproterenol increased plasma glucose in turkeys, but had no effect in chickens. 5. Phenylephrine increased plasma glucose after the bolus in turkeys but had no effect in chickens.
Asunto(s)
Glucemia/efectos de los fármacos , Catecolaminas/farmacología , Pollos/sangre , Corticosterona/farmacología , Pavos/sangre , Animales , Cromatografía Líquida de Alta Presión , Electroquímica , Isoproterenol/farmacología , Masculino , Fenilefrina/farmacología , Cloruro de Sodio/farmacologíaRESUMEN
The perfluorochemicals (PFC) have a high capacity for dissolving oxygen and carbon dioxide. Two experiments were conducted using PFC as diluent components to assess their effect on the fertilizing capacity of, and the hatchability of eggs from hens inseminated with, turkey semen stored for 30 min or 3 h. Two PFC were tested for the 30-min experiment: perfluorohexane (FC-72) and perfluorobutyltetrahydrofurane (FC-80). For the 3-h experiment, these PFC plus bis-1,2-(F-butyl)-ethene (F-44E) were studied. The PFC were emulsified with an equal volume of a standard aqueous salt diluent, Beltsville Poultry Semen Extender (BPSE). The BPSE alone was used as a control in both experiments. Semen was diluted 1:2 with oxygen-saturated extender and stored in closed tubes at 20 to 30 C prior to insemination. For the 30-min study, PFC diluents resulted in fertility rates comparable to (FC-72) or significantly greater than (FC-80) BPSE alone (P < or = .05). There was no significant difference in hatchability due to any semen treatment. When semen was stored for 3 h, PFC diluents resulted in comparable (FC-80) or significantly higher (F-44E and FC-72) fertility and hatchability rates. Significantly lower embryonic deaths at Day 10 of incubation were noted for all PFC diluents. These findings provide evidence that PFC emulsions are not toxic and prolong fertilizing capacity of turkey sperm during short-term storage, and suggest that providing oxygen carriers such as PFC in semen diluents may be beneficial.
Asunto(s)
Fertilización/efectos de los fármacos , Fluorocarburos/farmacología , Semen/fisiología , Animales , Femenino , Inseminación Artificial , Semen/efectos de los fármacos , PavosRESUMEN
Protease and basic amidase activity was found in the seminal plasma of the domestic turkey. Amidase activity, measured through use of N-alpha-benzoyl-DL-arginine-p-nitroanilide-HCL (BAPNA), was 23-28 times greater for turkey than for guinea fowl or chicken. Within the reproductive tract, seminal plasma from the vas deferens had much greater activity than testicular or epididymal fluids. Turkey seminal plasma enzyme (TSPE) purified by chromatography or isoelectric focusing showed three protein bands by PAGE, each resolving on SDS-PAGE into two subunits with molecular weights of approximately 28,000-32,000 and 38,000-44,000. One of the three proteins also contained a larger subunit (M(r) 76,000-81,000) thought to be transferrin. Turkey acrosin consisted of three subunits with molecular weights below 20,500. Acrosin, but not TSPE, was visualized in native gels with N-alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA)/Fast Garnet stain. Michaelis constants (BAPNA) for TSPE, acrosin, and trypsin were 2.41 +/- 0.12 x 10(-4) M (n = 5), 4.96-6.03 x 10(-4) M (n = 2), and 6.76 +/- 0.95 x 10(-4) M (n = 6), respectively. TSPE, like acrosin and trypsin, was inhibited by benzamidine but not iodoacetamide. While all natural trypsin inhibitors tested inhibited acrosin, TSPE was not inhibited by ovomucoid from chicken or turkey egg white.