... Under laboratory conditions the skin ordinarily becomes rough and tuberculated within one or ... more ... Under laboratory conditions the skin ordinarily becomes rough and tuberculated within one or two ... the skin of intact, laboratory-conditioned, red-spotted newts to become smooth and ... of 1-thyroxine had been added molted only rarely, but when thyroidectomized animals were put ...
Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the ... more Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the sea urchin, Lytechinus variegatus. To assay for NDA, isolated sperm heads were exposed to homogenate from mature unfertilized ova and then examined with phase-contrast optics. NDA is retained by dialysis and requires Ca++ at the time of homogenization as well as at the time of assay. Other divalent cations (Mg++, Sr++, Ba++, Mn++) can substitute for Ca++ but are much less effective. NDA is stable to cold (-20 degrees C, -76 degrees C) but not to heat (56 degrees C). The pH activity optimum is in the range 7-9. NDA is present in the supernatant after centrifugation at 150,000 g for 105 minutes. It elutes shortly following the void volume on a Sephacryl S-200 column, with an approximate M.W. of 100,000 daltons.
American journal of reproductive immunology and microbiology : AJRIM, 1986
The meaningful detection of antisperm antibody in immunologically infertile females has been conf... more The meaningful detection of antisperm antibody in immunologically infertile females has been confounded by the many methods of assay that exist. With many of these methods there is poor correlation of assay results with infertility. In this report, female rabbits were rendered partially or completely infertile by immunization with sperm fractions. A filter radioassay for antisperm antibody was developed that consists of incubating 10(7) sperm with sperm from immunized rabbits and 14C-Protein A, a long-lived and versatile indirect radiolabel for many antibodies of the IgG class. The spermatozoa are washed by rapid vacuum filtration on polycarbonate membrane filters instead of by time-consuming centrifugation. The filters with the collected spermatozoa are then counted in a liquid scintillation counter. Sera from female rabbits isoimmunized with sperm antigens show a highly significant correlation (r = -0.904; p less than 0.001) between assay results and infertility as measured by the...
ABSTRACT The effects of food restriction were investigated in adult male rats. Control animals we... more ABSTRACT The effects of food restriction were investigated in adult male rats. Control animals were fed ad libitum. Experimental animals were restricted to 15 or 5 grams/day. Parameters investigated included testis and body weight, testicular histology, mating rate, pregnancy rate, litter size, and early postnatal growth rate of offspring. Animals restricted to 15 grams/day were lighter than ad libitum controls. No other differences were observed between these two groups. Animals restricted to only 5 grams/day were not only lighter than the other two groups, they also had significantly lower testis weight, mating rate, pregnancy rate, and litter size. No histological differences in the testes were noted between groups. Offspring from all litters born gained weight in the first two days post-partum at the same rate, regardless of litter size.
In dedication to Dr. Frank Gwasdauskas, the rest of my committee, friends, family, faculty, and s... more In dedication to Dr. Frank Gwasdauskas, the rest of my committee, friends, family, faculty, and staff of Dairy Science Department who m all contributed in so many ways to make this an incredible experience, the gain of knowledge, and tremendous personal growth………. "What it takes to be a human being….. "If you take as your point of departure the human being and his relationship to the world as a human one, then you give love in return for love, trust in return for trust, etc... If you want to influence other people, then you have to be a person who genuinely stimulates and challenges others. Each of your relationships with people and with nature has to be a specific expression of your own true, individual life appropriate to the object of your intentions. If you love without awakening love in return, that is, if your love does not produce a loving person, if the expression of your being as a loving person does not make you loved, then your love is powerless and brings unhappiness."
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wast... more Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed I would like to thank all those involved in this research and without their help this
Received April 13, 1978) This investigation persues 2 goals: 1. to give data on the time course o... more Received April 13, 1978) This investigation persues 2 goals: 1. to give data on the time course of cleavage and blastocyst elevelopment in the cat, amI 2. to stuely the differentiation of trophoblast amI embryonic knot cells during cleavage and blastocyst formation in this species. Timed matings under standardized laboratory conditions arc used in order to obtain exactly elateel embryonic stages which are elescribed in detail. It is found that all preimplantation stages take comparatively long in the cat, i.e. roughly twice as long as in the rabbit; e.g. the embryos enter the uterus at 6 (- 7) days post coitum when they are in the morula 01' pre-blastocyst stage. After fixation in formol-alcohol-acetic acid which extracts certain fractions of proteins, it is found possible to distinguish, in cleavage stages, 2 categories of blastomeres which differ in den sity of remaining cytoplasmatic protein as weil as in cell polarity. Using serial sections anel com paring successive stage...
Page 1. Gamete Research 4:379-386 (1981) Immunoferritin Labeling of Rabbit Spermatozoa Steven M. ... more Page 1. Gamete Research 4:379-386 (1981) Immunoferritin Labeling of Rabbit Spermatozoa Steven M. Schrader, Ludeman A. Eng, and Charles B. Metz Institute for Molecular and Cellular Evolution, University of Miami, Coral Gables, Florida ...
Pig oocytes cultured at 39\s=deg\C had a higher percentage of polar body formation than did those... more Pig oocytes cultured at 39\s=deg\C had a higher percentage of polar body formation than did those cultured at 37\s=deg\C.A culture medium based on Medium 199 with Earle's salts and supplemented with 15% serum from a young castrated boar was just as good as the same formulation containing additional pyruvate, lactate and insulin, and superior to a modified Krebs\p=n-\Ringer bicarbonate medium. When the bicarbonate buffer system (Earle's salts) of Medium 199 was replaced with a phosphate buffer system (Hank's salts), the rate of polar body formation was decreased. When the Hank's based medium was supplemented with a bicarbonate buffer system, polar body formation was restored to the level in Earle's based medium. This suggests that CO2/bicarbonate may be important for the normal maturation of pig oocytes.
A procedure for the production of [14 C]proteln A is described wluch revolves reductlve methylatl... more A procedure for the production of [14 C]proteln A is described wluch revolves reductlve methylatlon of lysme residues with [X4C]formaldehyde and NaCNBH 3 The binding of [14C]protem A to IgG is apparently unaltered, as deternuned by competluve binding studies The use of [x4C]proteln A may be preferred to that of 125I-protein A when a radioactive label with a long half-life is desirable
Biochemical and Biophysical Research Communications, 1986
Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2... more Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2. Measurements of trypan blue exclusion indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of quin-2 loaded sperm was calculated to be 144 ± 14 nM. Spermatozoa capacitated in vitro either before or following quin-2 loading had intracellular free calcium levels similar to that of non-capacitated sperm. These studies indicate that the concentration of intracellular free calcium of ejaculated rabbit spermatozoa does not change as a result of in vitro capacitation.
Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=... more Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (r(2)=0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r(2)=0.20). PAF concentrations, incubation time and their interaction had a highly significant (P<0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the sperma...
The light microscopical morphology and proteinase activities are studied during the first phases ... more The light microscopical morphology and proteinase activities are studied during the first phases of implantation in the cat, i.e. at 12, 13 and 14 days post coitum (d.p.c.). Timed matings are used to obtain exact data on the time course of implantation events. Size and shape of the blastocysts and the topographical relationships between trophoblast, zona pellucida and endometrium are studied on cryostat sections. These observations indicate that the zona pellucida is being removed at 12 d p.c. by dissolution which starts at the abembryonic pole and lateral of the embryonic disc. Since the zona has, in spite of the considerable expansion of the blastocyst, a thickness of 8-10 ~tm at this stage, it must have undergone a process of swelling or material must have been added invisibly. Invasion of the trophoblast into the endometrium begins between 13 and 14 d p.c. and is fully under way at 14 d p.c. Widening of the glandular lumina in the neighborhood of the blastocysts at t2-13 d p.c. indicates an early preimplantation interaction between the blastocyst and the endometrium. Amino Acid Arylamidase (Aminopeptidase) activity is found, in histochemical tests, to be high in the trophoblast but low in the endometrium in all three investigated stages. Proteinase Activity is studied with a highly sensitive gelatin substrate film test. Moderate to medium activity is found in the trophoblast at 12-13 d p.c. Very high proteinase activity is present in the invasion zone at 14 d p.c. Experiments with a large number of specific proteinase inhibitors in vitro and preliminary investigation of pH dependence show that it is mainly due to a cathepsin-B-like endopeptidase. This enzyme can be traced to both the trophobiast and the uterine epithelium. The disintegrating zona pellucida shows, at i2 d p.c., only little gelatinolytic proteinase activity. A trypsin-like endopeptidase as described for the rabbit blastocyst could not be identified with certainty in the cat but
... Under laboratory conditions the skin ordinarily becomes rough and tuberculated within one or ... more ... Under laboratory conditions the skin ordinarily becomes rough and tuberculated within one or two ... the skin of intact, laboratory-conditioned, red-spotted newts to become smooth and ... of 1-thyroxine had been added molted only rarely, but when thyroidectomized animals were put ...
Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the ... more Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the sea urchin, Lytechinus variegatus. To assay for NDA, isolated sperm heads were exposed to homogenate from mature unfertilized ova and then examined with phase-contrast optics. NDA is retained by dialysis and requires Ca++ at the time of homogenization as well as at the time of assay. Other divalent cations (Mg++, Sr++, Ba++, Mn++) can substitute for Ca++ but are much less effective. NDA is stable to cold (-20 degrees C, -76 degrees C) but not to heat (56 degrees C). The pH activity optimum is in the range 7-9. NDA is present in the supernatant after centrifugation at 150,000 g for 105 minutes. It elutes shortly following the void volume on a Sephacryl S-200 column, with an approximate M.W. of 100,000 daltons.
American journal of reproductive immunology and microbiology : AJRIM, 1986
The meaningful detection of antisperm antibody in immunologically infertile females has been conf... more The meaningful detection of antisperm antibody in immunologically infertile females has been confounded by the many methods of assay that exist. With many of these methods there is poor correlation of assay results with infertility. In this report, female rabbits were rendered partially or completely infertile by immunization with sperm fractions. A filter radioassay for antisperm antibody was developed that consists of incubating 10(7) sperm with sperm from immunized rabbits and 14C-Protein A, a long-lived and versatile indirect radiolabel for many antibodies of the IgG class. The spermatozoa are washed by rapid vacuum filtration on polycarbonate membrane filters instead of by time-consuming centrifugation. The filters with the collected spermatozoa are then counted in a liquid scintillation counter. Sera from female rabbits isoimmunized with sperm antigens show a highly significant correlation (r = -0.904; p less than 0.001) between assay results and infertility as measured by the...
ABSTRACT The effects of food restriction were investigated in adult male rats. Control animals we... more ABSTRACT The effects of food restriction were investigated in adult male rats. Control animals were fed ad libitum. Experimental animals were restricted to 15 or 5 grams/day. Parameters investigated included testis and body weight, testicular histology, mating rate, pregnancy rate, litter size, and early postnatal growth rate of offspring. Animals restricted to 15 grams/day were lighter than ad libitum controls. No other differences were observed between these two groups. Animals restricted to only 5 grams/day were not only lighter than the other two groups, they also had significantly lower testis weight, mating rate, pregnancy rate, and litter size. No histological differences in the testes were noted between groups. Offspring from all litters born gained weight in the first two days post-partum at the same rate, regardless of litter size.
In dedication to Dr. Frank Gwasdauskas, the rest of my committee, friends, family, faculty, and s... more In dedication to Dr. Frank Gwasdauskas, the rest of my committee, friends, family, faculty, and staff of Dairy Science Department who m all contributed in so many ways to make this an incredible experience, the gain of knowledge, and tremendous personal growth………. "What it takes to be a human being….. "If you take as your point of departure the human being and his relationship to the world as a human one, then you give love in return for love, trust in return for trust, etc... If you want to influence other people, then you have to be a person who genuinely stimulates and challenges others. Each of your relationships with people and with nature has to be a specific expression of your own true, individual life appropriate to the object of your intentions. If you love without awakening love in return, that is, if your love does not produce a loving person, if the expression of your being as a loving person does not make you loved, then your love is powerless and brings unhappiness."
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wast... more Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed I would like to thank all those involved in this research and without their help this
Received April 13, 1978) This investigation persues 2 goals: 1. to give data on the time course o... more Received April 13, 1978) This investigation persues 2 goals: 1. to give data on the time course of cleavage and blastocyst elevelopment in the cat, amI 2. to stuely the differentiation of trophoblast amI embryonic knot cells during cleavage and blastocyst formation in this species. Timed matings under standardized laboratory conditions arc used in order to obtain exactly elateel embryonic stages which are elescribed in detail. It is found that all preimplantation stages take comparatively long in the cat, i.e. roughly twice as long as in the rabbit; e.g. the embryos enter the uterus at 6 (- 7) days post coitum when they are in the morula 01' pre-blastocyst stage. After fixation in formol-alcohol-acetic acid which extracts certain fractions of proteins, it is found possible to distinguish, in cleavage stages, 2 categories of blastomeres which differ in den sity of remaining cytoplasmatic protein as weil as in cell polarity. Using serial sections anel com paring successive stage...
Page 1. Gamete Research 4:379-386 (1981) Immunoferritin Labeling of Rabbit Spermatozoa Steven M. ... more Page 1. Gamete Research 4:379-386 (1981) Immunoferritin Labeling of Rabbit Spermatozoa Steven M. Schrader, Ludeman A. Eng, and Charles B. Metz Institute for Molecular and Cellular Evolution, University of Miami, Coral Gables, Florida ...
Pig oocytes cultured at 39\s=deg\C had a higher percentage of polar body formation than did those... more Pig oocytes cultured at 39\s=deg\C had a higher percentage of polar body formation than did those cultured at 37\s=deg\C.A culture medium based on Medium 199 with Earle's salts and supplemented with 15% serum from a young castrated boar was just as good as the same formulation containing additional pyruvate, lactate and insulin, and superior to a modified Krebs\p=n-\Ringer bicarbonate medium. When the bicarbonate buffer system (Earle's salts) of Medium 199 was replaced with a phosphate buffer system (Hank's salts), the rate of polar body formation was decreased. When the Hank's based medium was supplemented with a bicarbonate buffer system, polar body formation was restored to the level in Earle's based medium. This suggests that CO2/bicarbonate may be important for the normal maturation of pig oocytes.
A procedure for the production of [14 C]proteln A is described wluch revolves reductlve methylatl... more A procedure for the production of [14 C]proteln A is described wluch revolves reductlve methylatlon of lysme residues with [X4C]formaldehyde and NaCNBH 3 The binding of [14C]protem A to IgG is apparently unaltered, as deternuned by competluve binding studies The use of [x4C]proteln A may be preferred to that of 125I-protein A when a radioactive label with a long half-life is desirable
Biochemical and Biophysical Research Communications, 1986
Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2... more Ejaculated rabbit spermatozoa were loaded with the calcium-selective fluorescent indicator quin-2. Measurements of trypan blue exclusion indicated that cell viability was not affected by quin-2 loading. The concentration of intracellular free calcium of quin-2 loaded sperm was calculated to be 144 ± 14 nM. Spermatozoa capacitated in vitro either before or following quin-2 loading had intracellular free calcium levels similar to that of non-capacitated sperm. These studies indicate that the concentration of intracellular free calcium of ejaculated rabbit spermatozoa does not change as a result of in vitro capacitation.
Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=... more Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (r(2)=0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r(2)=0.20). PAF concentrations, incubation time and their interaction had a highly significant (P<0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the sperma...
The light microscopical morphology and proteinase activities are studied during the first phases ... more The light microscopical morphology and proteinase activities are studied during the first phases of implantation in the cat, i.e. at 12, 13 and 14 days post coitum (d.p.c.). Timed matings are used to obtain exact data on the time course of implantation events. Size and shape of the blastocysts and the topographical relationships between trophoblast, zona pellucida and endometrium are studied on cryostat sections. These observations indicate that the zona pellucida is being removed at 12 d p.c. by dissolution which starts at the abembryonic pole and lateral of the embryonic disc. Since the zona has, in spite of the considerable expansion of the blastocyst, a thickness of 8-10 ~tm at this stage, it must have undergone a process of swelling or material must have been added invisibly. Invasion of the trophoblast into the endometrium begins between 13 and 14 d p.c. and is fully under way at 14 d p.c. Widening of the glandular lumina in the neighborhood of the blastocysts at t2-13 d p.c. indicates an early preimplantation interaction between the blastocyst and the endometrium. Amino Acid Arylamidase (Aminopeptidase) activity is found, in histochemical tests, to be high in the trophoblast but low in the endometrium in all three investigated stages. Proteinase Activity is studied with a highly sensitive gelatin substrate film test. Moderate to medium activity is found in the trophoblast at 12-13 d p.c. Very high proteinase activity is present in the invasion zone at 14 d p.c. Experiments with a large number of specific proteinase inhibitors in vitro and preliminary investigation of pH dependence show that it is mainly due to a cathepsin-B-like endopeptidase. This enzyme can be traced to both the trophobiast and the uterine epithelium. The disintegrating zona pellucida shows, at i2 d p.c., only little gelatinolytic proteinase activity. A trypsin-like endopeptidase as described for the rabbit blastocyst could not be identified with certainty in the cat but
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