Effects of high copper supplements on performance, health, plasma copper and enzymes in goats.

Six growing female Nubian goats (average BW=34.8+/-0.55kg, 7-8 months of age) were randomly assigned to either a basal diet (BD, 10-15ppm Cu/DM), or to medium Cu (MC, BD+50mgCu), or to high Cu (HC, BD+100mgCu) diets for 9 weeks. This level would cause Cu toxicity in sheep, but none occurred in the goats. Therefore, Cu supplementation was then increased to 150 and 300mg per head per day, for the following 14 weeks; to 300 and 600mg per head per day, for the next 8 weeks; and to 600 and 1200mg per head per day, for an additional 4 weeks, in the MC and HC group, respectively. Body weight and vital signs were recorded and blood samples collected at different time intervals. Hematological parameters, plasma Cu, sorbitol dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), and gamma-glutamyl transferase (GGT) were determined. At the termination of the study, tissue Cu concentration in different organs was also determined. During first 23 weeks (<300mgCu per day) of the study there were no apparent signs of Cu toxicity. Cu supplementation at 600mg per head per day in young Nubian does, had no effect on respiration rate (RR), heart rate (HR), and decreased (P<0.05) rectal temperature (RT) in the HC group only. Leukocyte counts were positively correlated with Cu supplementation (r=+0.296, P<0.02) and negatively correlated (r=-0.254, P<0.05) with RT in the HC group. Plasma SDH increased (P<0.05) when Cu supplementation was >/=300mg per head per day, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Increases (P<0.05) in GOT were noted when Cu intake was >/=600mg per head per day. Contrary to the results observed for SDH and GOT, feeding goats 50mgCu per day or more, resulted in an increased plasma GGT as compared to BD goats. Levels of SDH, GOT and GGT of the BD goats were within normal range. Plasma Cu was not indicative of Cu status of animals. Copper improved ADG by 28% at the 100-150ppm level in diet. No relationship between Cu intake and hair Cu was found in the present study. Highest concentration of Cu was found in liver, followed by duodenum, rumen and brain. Results of this study indicate that goats are more resistant to Cu toxicity than sheep. This is one of the first reports documenting significant differences in Cu requirements and tolerance between goats and sheep.

Postnatal differentiation of the ductus deferens, tail of the epididymis, and distal body of the epididymis in goats occurs independently of rete testis fluid.

Observations from extratesticular rete-ligated, mature goats indicated that epithelial morphology in the tail of the epididymis can be maintained without any input from testicular fluid (Goyal et al., Acta Anat., 1994;150: 127-135). Hence, the objective of this study was to determine whether the tail of the epididymis and/or other regions of the male excurrent ducts can differentiate prior to the appearance of lumen in the seminiferous tubules, which is an indicator for the onset of seminiferous tubular fluid secretion. Based on age and scrotal circumference (SC), 20 male goats were divided into four groups of five animals each: 1-4 weeks (SC, 6.5-7.5 cm), 7-10 weeks (SC, 8.5-11.0 cm), 12-15 weeks (SC, 11.0-14.0 cm), and 15-25 weeks (SC, 16.0-19.0 cm). Tissues were collected from the testis, six regions of the epididymis (proximal, middle and distal head; proximal and distal body; and tail), and the ductus deferens, and were processed for light and electron microscopic examination. Changes in epithelial height and cytological features associated with absorption (microvilli, pinocytotic and coated vesicles) and protein secretion (RER, Golgi body) were used as markers for differentiation. Differentiation of all of these features was comparable to that observed in the 15-25-week-old animals in the ductus deferens by > or = 1 week, in the tail of the epididymis by > or = 7 weeks, in the distal body of the epididymis by > or = 12 weeks, and in the proximal body of the epididymis and all three regions of the head of the epididymis by > or = 15 weeks. Seminiferous tubules developed lumens between 12 and 15 weeks. In conclusion, epithelial differentiation in the ductus deferens, tail of the epididymis, and distal body of the epididymis follows a time-dependent, spatial, ascending order and is achieved before lumen formation in the seminiferous tubules. Conversely, epithelial differentiation in all three regions of the head and the proximal body of the epididymis occurs simultaneously and after lumen formation in the seminiferous tubules.

Effects of androgen deprivation in the goat epididymis.

With the deprivation of both circulating androgen (CA) and luminal androgen (LA; orchiectomized goats), the epithelial height (EH) in regions I-IV of the epididymis was reduced to 28, 67, 58 and 56% of that of controls, respectively, but it was increased to 109% of that of controls in region V. Similarly, the volume density of epithelium (VDE) in regions I-V was reduced to 33, 49, 45, 41 and 70% of that of controls, respectively. Conversely, in the absence of LA only (extratesticular-rete-ligated goats), while both EH and VDE were reduced to almost 50% of those of controls in region I, they remained similar to those of controls in other regions. The morphological changes in the epithelium such as cytoplasmic regression, loss of stereocilia and disorderly arrangement of epithelial cells were maximal in region I, moderate in regions II-IV and minimal in region V. Testosterone treatment appreciably reduced the degenerative changes caused by orchiectomy in all regions except region I where the restorations were marginal at best. Hence, the results suggest a differential epididymal response to androgen deprivation. Whereas the LA and/or other rete fluid components seem essential for maintaining the epithelial structure of region I, the CA alone can maintain, at least partially, the epithelial structure of regions II-IV and almost completely that of region V.

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control.

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38 degrees C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38 degrees C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.

Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chlamydomonas reinhardtii y-1 at 38 degrees C.

The initial kinetics of accumulation of chlorophylls (Chl) were analyzed during optimal greening of Chlamydomonas reinhardtii y-1 at 38 degrees C. Acetate was required for maximal synthesis of Chl, which occurred at a linear rate when degreened cells were exposed to light. During the first hour Chl a and b accumulated predominantly as geranylgeraniol esters, with lesser amounts of the species with more reduced alcohol side chains. When Chl synthesis was blocked either by treatment with gabaculine or by transfer to the dark, the distribution shifted to the more reduced forms. Similar kinetic patterns indicated that a common pool of chlorophyllides a and b provided substrate for the enzymatic system that performs esterification and reduction of the sldechain for each group of Chl. Chl b was essentially quantitatively integrated into light-harvesting complexes as indicated by energy transfer to Chl a. In the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis, Chl b did not accumulate and Chl a production was reduced about one-half. The results demonstrate that Chl a/b-protein complexes assemble rapidly during greening and that reduction of the alcohol side chain of the Chl is not required for assembly of these complexes.

Effects of age and season on the type and occurrence of sperm abnormalities in Nubian bucks.

Effects of age and season on type and occurrence of sperm abnormalities were examined in semen samples collected from 3 groups of Nubian bucks at ages of 4 to 9 months, 10 to 21 months, and 39 to 50 months. The average total percentage of sperm abnormalities at the onset of puberty (141 +/- 4 days) was 64.6 +/- 14.8% (head, 19.5 +/- 13.6%; middle piece, 17.2 +/- 9.3%; and proximal protoplasmic droplets, 14.6 +/- 10.5%), but this improved rapidly and was reduced to 12.5 +/- 7.5% by 8 months of age (head, 1.9 +/- 4.5%; middle piece, 4.6 +/- 2.8%). Further increase in age, at least up to 4 years, did not reveal a significant effect (P less than 0.05) on the type or percentage of total abnormalities. Similar to age, a comparison of data among seasons did not reveal a significant effect on the type or occurrence of sperm abnormalities in 10- to 21-month-old or 39- to 50-month-old bucks. Seemingly, Nubian bucks started producing good quality semen at 8 months of age, and season did not influence sperm abnormalities.

Characterization of rabbit stromal fibroblasts derived from red and yellow bone marrow.

Rabbit stromal fibroblasts subcultured from red and yellow bone marrow and implanted beneath the renal capsule form ossicles the hemic cellularity of which mirrors the cellularity of the marrow used for culture. Although the cultured red and yellow marrow cells are similar in fine-structural appearance, they differ strikingly in enzymatic content of alpha-naphthylbutyrate esterase, which is abundant only in the cells derived from yellow marrow. Other observers (20, 21) have proposed that stromal fibroblasts are preadipocytes, and this data suggests that those derived from yellow marrow have the phenotype of more differentiated adipocytes. On the other hand, fibroblasts derived from red and yellow bone marrow show no differences in their profiles of procollagen synthesis. Both types of fibroblasts secrete type III procollagen as the major species, with a I/III ratio of 1:3; in contrast, rabbit dermal fibroblasts have a prominent peak of type I procollagen. The similarity of stromal cells derived from red and yellow bone marrow in procollagen synthesis suggests that the collagen part of the extracellular matrix is not the only basis for their intrinsic difference in capacity for hematopoiesis.

The question of bone marrow stromal fibroblast traffic.

Bone marrow stromal fibroblasts (CFU-F) normally do not exchange bone marrow sites in vivo. Restitution of the CFU-F after radiation damage is primarily recovery by the local fibroblasts from potentially lethal damage. Migration of stromal fibroblasts from shielded sites to an irradiated site makes a minimal contribution, if any, to CFU-F recovery. Determination of the relative contribution of donor stromal cells in bone marrow transplants by karyotyping the proliferating bone marrow stromal cells in vitro may not reflect the relative distribution of fibroblasts in the marrow. If there is residual damage to the host stromal fibroblasts from treatment before transplantation, these cells may not be able to proliferate in vitro. Therefore, an occasional transplanted fibroblast may contribute most of the metaphase figures scored for karyotype.

Decrease in hematopoietic stem cell domains as a delayed effect of x-irradiation.

Although the hematopoietic integrity of locally X-irradiated sites can be restored for a time even after fairly large doses, a secondary aplasia often occurs some months later. To gain further insight into this delayed effect within the framework of the stem cell regulatory domain hypothesis, we characterized the growth kinetics of spleen colony forming units (CFU-S) in WBB6FI-+/+ bone marrow transplanted into WBB6FI-W/WV mice in which one leg had been exposed to 10-30 Gy of X rays 4-5 months previously. Compared to unirradiated contralateral marrow, fewer CFU-S either reached the previously irradiated marrow or were seeded into sites that could support growth. The initial exponential growth of effectively seeded CFU-S was unchanged, but growth deceleration (inflection point) occurred at a lower level of CFU-S in marrow previously irradiated with 20-30 Gy. This change in the inflection point indicates a radiation dose-dependent decrease consistent with the decrease in bone marrow cellularity. The decrease in effective stem cell domains after 20 Gy was calculated to be about 35%. We interpret these results to reflect the highly localized nature of delayed radiation damage to the marrow microenvironment.

Hematopoietic microenvironment transfer by stromal fibroblasts derived from bone marrow varying in cellularity.

Autologous fibroblast derivatives of red and yellow marrow of rabbits were shown to differ in their capability to transfer a hematopoietic microenvironment upon implantation under the renal capsule. Although a heterotopic ossicle formed in each instance, the quality of the associated medullary tissue mirrored the quality of the bone marrow used to generate the stromal fibroblasts. Thus, fibroblasts cultured from a cellular marrow produced a stroma with numerous hematopoietic foci whereas those cultured from a severely hypocellular marrow produced a stroma with mainly fat cells. The results with 21 implants point to a transmittable regulatory role of a class of stromal fibroblasts.

Partitioning of bone marrow into stem cell regulatory domains.

To examine the hypothesis that bone marrow consists of discrete stem cell regulatory volumes or domains, we studied spleen colony-forming unit (CFU-S) population growth kinetics in unirradiated WBB6F1-W/Wv mice receiving various doses of +/+ bone marrow cells. Assay of femoral marrow CFU-S content in the eight recipient dose groups revealed a family of growth curves having an initial dose-independent exponential phase and a subsequent dose-dependent deceleration phase. CFU-S content at the growth transition (inflection point) was not a simple linear function of inoculum dose but was shown rather to reflect a random distribution of initially seeded donor CFU-S in discrete volumes of recipient bone marrow. The inoculum dose resulting in a mean of 1 CFU-S per bone marrow sampling unit was estimated to be 17 x 10(6) bone marrow cells, corresponding to a total marrow uptake of approximately 5100 CFU-S (based on a seeding efficiency factor of 10%). If we assume single-hit kinetics, it follows that the recipient W/Wv bone marrow may contain approximately 5100 domains in which stem cell proliferation is geared to the density of the stem cell population. When the various inocula were corrected for multiple seeding in a given domain, the mean inflection point per domain was similar and indicative of five or so divisions before departure from exponential growth at approximately 20% of final CFU-S content 8 days after bone marrow injection. The partitioning of bone marrow into highly localized functional units is consistent with the putative regulatory role of short-range interactions between stem cells and essential stromal elements.

Hematopoietic stem cell proliferative behavior as revealed by bromodeoxyuridine labeling.

Hematopoietic stem cells (CFUS) are thought to represent a heterogeneous population with different probabilities of self-renewal and different rates of proliferation. As an approach to further characterization of this population, we determined the disappearance of bromodeoxyuridine (BrdUrd)-induced CFUS sensitization to ultraviolet light in normal mice infused with BrdUrd for three weeks and in hydroxyurea-pretreated mice regenerating their CFUS pool during a one-week BrdUrd infusion. the same exponential disappearance with a T1/2 of six days was found in each case. From this and from the apparent absence of a secondary slope on the sensitization decay curve for the recovered hydroxyurea-treated bone marrow, we conclude that fewer than 10% of CFUS may be mitotically quiescent for prolonged periods and that the age structure of the CFUS population reflects a relatively short proliferative history.

Turnover of circulating hematopoietic stem cells.

Short-term parabiosis of male and female CBA/CaJ mice was used to investigate the turnover of circulating hematopoietic stem cells. The exchange and subsequent disappearance of donor stem cells were monitored by spleen colony assay and chromosome analysis of individual colonies. The results revealed an exponential disappearance of pluripotent stem cells from blood with a characteristic half time of 1.7 h. Blood-borne stem cells were shown to be equilibrated with a subpopulation of marrow stem cells exhibiting a disappearance half time of 9.5 h. Splenectomy did not change the apparent rate of stem cell removal from the blood.

Differential response of early erythropoietic and granulopoietic progenitors to dexamethasone and cortisone.

The sensitivity of erythropoietic (BFU-E) and granulopoietic (CFU-C) progenitor cells to dexamethasone and cortisone was studied in cultures of mouse bone marrow. Although the log dose-response relationships had a similar form, the BFU-E were much more sensitive than the CFU-C to either glucocorticoid. The dexamethasone concentration for 50% inhibition was 3 X 10)-9) M for BFU-E and 60 X 10(-9) M for CFU-C. The differential sensitivity to cortisone was even greater, with 60% inhibition of BFU-E and 18% inhibition of CFU-C at 0.1 microgram/ml. These findings suggest a specific rather than a general response to glucocorticoids and indicate that granulocyte-macrophage progenitors are less affected than early erythroid progenitors by physiologic concentrations of these hormones.

Hematopoietic stem cell regulatory volumes as revealed in studies of the bgj/bgj:W/WV chimera.

The kinetics of bone marrow replacement was studied in W/WV mice implanted with gbj/bgj (beige) stem cells, with the characteristic beige neutrophil marker as a criterion of the takeover of host marrow by donor marrow. A hyperbolic pattern of W/WV marrow replacement conforming to a log dose-response was observed in experiments encompassing a 50-fold range of bgj/bgj inoculum doses and a 2-yr period of observation. The dose-response relationships were consistent with random seeding of stem cells in the host marrow coupled with a decreasing efficiency of secondary colonization by local migration. Application of single-hit Poisson sampling statistics to the dose-response data led to the hypothesis that mouse bone marrow is compartmentalized into essentially self-contained stem cell regulatory volumes or domains. We estimate that W/WV marrow contains about 2,600 stem cells regulatory units with an average volume of about 10(8) micron3, a dimension consistent with the presumptive role of short-range cell-cell interactions in the regulation of pluripotent stem cells. Our analysis of the dose-response data is also indicative of the discontinuous and limited nature of local stem cell migration in a cellular marrow, a consideration that may be of practical as well as theoretical interest.

Concentration gradient of blood stem cells in mouse bone marrow--an open question.

The distribution of pluripotential blood stem cells (CFU-S) in bone marrow was studied in four strains of mice. Analyses of paired samples containing various fractions of axial and marginal bone marrow cells, as well as longitudinal 200-micrometer sections, revealed a rather uniform spatial distribution of CFU-S across the diameter of the femoral medullary cavity, in contrast to the finding by others of a CFU-S concentration gradient extending from the endosteum to the central longitudinal axis of bone marrow. The existence of a stem cell gradient is therefore open to question.

Marrow stem cell release in the autorepopulation assay.

The early migration of stem cells from a shielded marrow to an irradiated spleen has been re-evaluated, and the findings have been compared with the results of earlier studies. The composite data reveal a constant rate during the first 24 h after irradiation, with a slope of 1.6 cells per h and an intercept of 2.4. The positive intercept is interpreted to signify an immediate brief perturbation of CFU's release. The low concentration of CFUs in the bloodstream, despite their continuous migration from the shielded marrow, is indicative of a rapid, and probably greatly increased, blood turnover. Despite the constancy of stem cell seeding, it is not yet possible to determine whether the rate of stem cell release is different in shielded marrow than in normal marrow. The resolution of this question requires more precise information about spleen seeding efficiency in the autorepopulation assay and about the normal turnover rate of stem cells in the bloodstream.