Results from demineralized bone creep tests suggest that collagen is responsible for the creep behavior of bone

Cortical and trabecular bone have similar creep behaviors that have been described by power-law relationships, with increases in temperature resulting in faster creep damage accumulation according to the usual Arrhenius (damage rate approximately exp (-Temp.-1)) relationship. In an attempt to determine the phase (collagen or hydroxyapatite) responsible for these similar creep behaviors, we investigated the creep behavior of demineralized cortical bone, recognizing that the organic (i.e., demineralized) matrix of both cortical and trabecular bone is composed primarily of type I collagen. We prepared waisted specimens of bovine cortical bone and demineralized them according to an established protocol. Creep tests were conducted on 18 specimens at various normalized stresses sigma/E0 and temperatures using a noninvasive optical technique to measure strain. Denaturation tests were also conducted to investigate the effect of temperature on the structure of demineralized bone. The creep behavior was characterized by the three classical stages of decreasing, constant, and increasing creep rates at all applied normalized stresses and temperatures. Strong (r2 > 0.79) and significant (p < 0.01) power-law relationships were found between the damage accumulation parameters (steady-state creep rate d epsilon/dt and time-to-failure tf) and the applied normalized stress sigma/E0. The creep behavior was also a function of temperature, following an Arrhenius creep relationship with an activation energy Q = 113 kJ/mole, within the range of activation energies for cortical (44 kJ/mole) and trabecular (136 kJ/mole) bone. The denaturation behavior was characterized by axial shrinkage at temperatures greater than approximately 56 degrees C. Lastly an analysis of covariance (ANCOVA) of our demineralized cortical bone regressions with those found in the literature for cortical and trabecular bone indicates than all three tissues creep with the same power-law exponents. These similar creep activation energies and exponents suggest that collagen is the phase responsible for creep in bone.     

Heat resistance of allograft tissue

Lyophilized allograft musculoskeletal tissue is generally intended to be stored at “ambient” or “room” temperature, and usually is kept in climate controlled indoor storage areas. However, there is a question of what temperature extremes tissue may see, especially during transportation, in that these extremes may exceed even the limits of “ambient” conditions. Temperatures may become quite hot, but only for a few hours and only during daytime. Damage from high temperatures, if it occurs, is expected to be evident by damage to the collagen component of bone, soft tissue, and demineralized bone, as well as to the growth factors contained in demineralized bone. If damage is significant, then temperature monitoring requirements for lyophilized allograft tissue might be necessary. To answer this question, a literature review was carried out to look at the short-term temperature resistance of collagen and demineralized bone. Both collagen and the growth factors in demineralized bone show remarkable short term tolerance to elevated temperatures in the dry state, and it was concluded that temperature excursions of 50°C or less, lasting for a few days or less, would not cause any significant deterioration. This means that temperature monitoring also should not be required.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Nanosecond fluctuations of the molecular backbone of collagen in hard and soft tissues: a carbon-13 nuclear magnetic resonance relaxation study

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis

It has long been known that core body temperature declines with age, with temperatures of 35.5°C or below common in the elderly. However, the effects of temperature reduction on bone cell function and skeletal homeostasis have been little studied. We investigated the effects of mild hypothermia (35.5°C) and severe hypothermia (34°C) on bone-forming osteoblasts, and bone-resorbing osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by 75% at 35.5°C and by 95% at 34°C after 14-16 days culture, compared to 37°C. In addition to reductions in osteoblast cell number, expression of mRNAs for Runx2, alkaline phosphatase, osteocalcin and type I collagen were also down-regulated in hypothermia. In contrast, formation of osteoclasts in mononuclear cell cultures derived from mouse marrow, showed a 1.5 to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Taken together, these data show that hypothermia exerts reciprocal effects on bone cell function by retarding osteoblast differentiation and bone formation, whilst increasing osteoclastogenesis and thus resorption. These results suggest the possibility that hypothermia in the elderly could potentially have a direct, negative impact on bone metabolism.     

Thermal stabilization of collagen molecules in bone tissue

Differential thermal calorimetry (DSC) analysis of partially dehydrated bovine bone, demineralized bone and bovine tendon collagen was performed up to 300 °C to determine factors influencing stability of mineralized collagen in bone tissue. Two endothermal regions were recognized. The first, attributed to denaturation of collagen triple helix, was hydration dependent and had a peak at 155–165 °C in bone, 118–137 °C in tendon and 131–136 °C in demineralized bone. The second region extended from 245 to 290 °C in bone and from 200 to 280 °C in tendon and was connected with melting and decomposition of collagen. Differences in thermodynamic parameters between cortical and trabecular bone tissue were stated. Activation energy of collagen unfolding in native bone tissue increased with dehydration of the bone. From the results of the present study we conclude that dehydrated bone collagen is thermally very stable both in native and in demineralized bone. Presence of mineral additionally stabilizes bone tissue.     

Technical note: Examining the use of ethylenediaminetetraacetic acid for humic extraction of ancient bone

We examined the efficacy of ethylenediaminetetraacetic acid (EDTA) for removing humic contaminants from collagen extracted from ancient bone. Humic contaminants must be removed to obtain reliable stable isotope values from ancient bone collagen, given that humic acids have consistently lower δ¹³C values than collagen. The purpose of our research was to examine if EDTA treatment could effectively remove humic contaminants from bone collagen and thus serve as an alternative to the commonly implemented sodium hydroxide (NaOH) treatment, which may be associated with large collagen losses in poorly preserved samples. We compared the isotopic and elemental composition of ancient samples treated with EDTA alone, samples demineralized in hydrochloric acid (HCl) and rinsed in EDTA, samples treated with HCl alone, and samples demineralized in HCl and rinsed with NaOH. The samples used in the analyses were selected because they presented evidence of substantial humic contamination. We found that NaOH was the most effective agent for reducing humic contaminants as evidenced by the samples treated with this agent having higher δ¹³C values and lower C:N ratios relative to other treatments. The results from samples treated with EDTA suggest that this chemical cannot effectively remove humic contaminants given that these samples had significantly higher C:N ratios and lower δ¹³C and δ¹⁵N values relative to the HCl/NaOH treatment. Our results demonstrate that when performing stable isotope analysis of ancient bone collagen suspected to be contaminated with humic acids, NaOH is the most effective chemical agent to remove humic contaminants, while EDTA cannot perform this task.     

Characterization of primary thermal degradation features of lignocellulosic biomass after removal of inorganic metals by diverse solvents

Poplar wood powders were treated with distilled water, tap water, HCl and HF, respectively, to remove inorganics from the biomass and to investigate effect of demineralization processes on pyrolysis behavior of the biomass. TG and DTG revealed that maximum degradation temperatures rose slightly from 362°C for control to 372°C, 366°C and 368°C after demineralization with distilled water, HCl and HF, respectively. Maximum degradation rates also increased from 0.96%/°C for control to 1.15%/°C for HF-biomass, 1.23%/°C for DI-H(2)O-biomass, and 1.55%/°C for HCl-biomass. Analytical pyrolysis-GC/MS of demineralized biomasses produced approximately 45 pyrolysis compounds. Total amount of low molecular weight compounds, such as acetic acid, acetol, and 3-hydroxypropanal, was significantly lowered in the demineralized biomasses. But levoglucosan increased 2-10-folds in the demineralized biomasses. One of the features regarding lignin derivatives was the reduction of the amount of C6-type phenols, such as phenol, guaiacol, and syringol after demineralization.     

Thermal stabilization of collagen in skin and decalcified bone

The state of collagen molecules in the fibres of tail tendon, skin and demineralized bone has been investigated in situ using differential scanning calorimetry (DSC). Hydroxyproline analysis and tissue digestion with bacterial collagenase and trypsin were used to confirm that the common cause of all the DSC endotherms was collagen denaturation. This occurred within a narrow temperature range in tendons, but over a wide temperature range in demineralized bone and old skin and demonstrated that in tendon and demineralized bone at least the same type I collagen molecule exists in different thermal states. Hypothesizing that this might be caused by different degrees of confinement within the fibre lattice, experiments were performed to measure the effect of changing the lattice dimensions by extracting the collagen into dilute solution with pepsin, swelling the lattice in acetic acid, and contracting the lattice by dehydration. A theoretical analysis was undertaken to predict the effect of dehydration. Results were consistent with the hypothesis, demonstrating that collagen molecules within the natural fibres of bone and old skin are located at different intermolecular spacings, revealing differences between molecules in the magnitude of either the attractive or repulsive forces controlling their separation. One potential cause of such variation is known differences in covalent cross-linking.     

Effect of temperature on low-strength wastewater treatment by UASB reactor using poly(vinyl alcohol)-gel carrier

The feasibility of treating low-strength wastewater with an up-flow anaerobic sludge blanket (UASB) reactor, using a poly(vinyl alcohol)-gel carrier, at various temperatures and hydraulic retention times (HRTs) was examined. The temperature was decreased from 35°C to 25°C and then to 15°C. The HRT was reduced from 2.0 h to 0.22 h. The COD removal rate reached 28 kg-COD m(-3)d(-1) at 35°C, 16 kg-COD m(-3)d(-1) at 25°C, and 6 kg-COD m(-3)d(-1) at 15°C. The COD removal rate was reduced by half for each temperature reduction of 10°C.     

Survival of Campylobacter jejuni in chicken meat at frozen storage temperatures

The aim of this study was to determine the survival of Campylobacter jejuni in chicken meat samples at frozen temperatures and given length of incubation and to determine the impact of aerobic bacteria on the survival of C. jejuni. The chicken meat samples were inoculated with C. jejuni NCTC 11351 suspensions and stored in bags at temperatures of -20°C and -70°C. The mean value of C. jejuni from meat samples decreased from 7.52 log10 CFU/g after 30 minutes of incubation at ambient temperature, to 3.87 log10 CFU/g on the eighth week of incubation at -20°C, and to 3.78 log10 CFU/g at incubation at -70°C after the same incubation period. Both freezing temperatures, -20°C and -70°C, decreased the number of campylobacters. The presence of aerobic mesophilic bacteria did not influence the survival of C. jejuni in chicken meet samples. Keeping poultry meat at freezing temperatures is important for the reduction of C. jejuni, which has a strong influence on the prevention of occurrence of campylobacteriosis in humans.     

Effects of perturbed geomagnetic field on connective tissue metabolism and some systems of homeostasis after hydroxyapatite implantation

The influence of perturbed geomagnetic field (PGMF) on glucocorticoid, epinephrine, hydroxyproline content in intact rats and after intramuscular implantation of demineralized bone matrix and combined (hydroxyapatite/bone meal) samples was investigated. It was found that the exposure of the intact rats to PGMF led to decrease in free and peptide-bound hydroxyproline in plasma. In the rats with hydroxyapatite/bone meal samples placed in ectopic sites the exposure to PGMF normalized hydroxyproline, glucocorticoid and epinephrine content.     

Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats

The compounds resulting from the reaction of glucose with proteins (advanced glycation products) can be important markers of chronic diabetic complications. To test the possible diagnostic value of advanced glycation products containing the furoyl moiety, collagen samples from diabetic and healthy rats were analyzed by parent ion spectroscopy. In our study, we compared normal collagen, diabetic collagen and normal collagen incubated with different glucose concentrations and we employed different hydrolysis procedures (HCl and proteinase). Mass spectroscopic measurements performed on hydrolyzed samples showed that either different samples or different hydrolysis procedures produce a similar set of furoyl-containing compounds. 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) which has been reported to be one of the advanced glycation products, was never found in any of the samples examined. Hence neither FFI nor furoyl-containing molecules can be considered markers of advanced glycation processes.     

Photosynthetic and respiratory acclimation and growth response of Antarctic vascular plants to contrasting temperature regimes

Air temperatures have risen over the past 50 yr along the Antarctic Peninsula, and it is unclear what impact this is having on Antarctic plants. We examined the growth response of the Antarctic vascular plants Colobanthus quitensis (Caryophyllaceae) and Deschampsia antarctica (Poaceae) to temperature and also assessed their ability for thermal acclimation, in terms of whole-canopy net photosynthesis (P(n)) and dark respiration (R(d)), by growing plants for 90 d under three contrasting temperature regimes: 7°C day/7°C night, 12°C day/7°C night, and 20°C day/7°C night (18 h/6 h). These daytime temperatures represent suboptimal (7°C), near-optimal (12°C), and supraoptimal (20°C) temperatures for P(n) based on field measurements at the collection site near Palmer Station along the west coast of the Antarctic Peninsula. Plants of both species grown at a daytime temperature of 20°C had greater RGR (relative growth rate) and produced 2.2-3.3 times as much total biomass as plants grown at daytime temperatures of 12° or 7°C. Plants grown at 20°C also produced 2.0-4.1 times as many leaves, 3.4-5.5 times as much total leaf area, and had 1.5-1.6 times the LAR (leaf area ratio; leaf area:total biomass) and 1.1-1.4 times the LMR (leaf mass ratio; leaf mass:total biomass) of plants grown at 12° or 7°C. Greater RGR and biomass production at 20°C appeared primarily due to greater biomass allocation to leaf production in these plants. Rates of P(n) (leaf-area basis), when measured at their respective daytime growth temperatures, were highest in plants grown at 12°C, and rates of plants grown at 20°C were only 58 (C. quitensis) or 64% (D. antarctica) of the rates in plants grown at 12°C. Thus, lower P(n) per leaf area in plants grown at 20°C was more than offset by much greater leaf-area production. Rates of whole-canopy P(n) (per plant), when measured at their respective daytime growth temperatures, were highest in plants grown at 20°C, and appeared well correlated with differences in RGR and total biomass among treatments. Colobanthus quitensis exhibited only a slight ability for relative acclimation of P(n) (leaf-area basis) as the optimal temperature for P(n) increased from 8.4° to 10.3° to 11.5°C as daytime growth temperatures increased from 7° to 12° to 20°C. There was no evidence for relative acclimation of P(n) in D. antarctica, as plants grown at all three temperature regimes had a similar optimal temperature (10°C) for P(n). There was no evidence for absolute acclimation of P(n) in either species, as rates of P(n) in plants grown at a daytime temperature of 12°C were higher than those of plants grown at daytime temperatures of 7° or 20°C, when measured at their respective growth temperatures. The poor ability for photosynthetic acclimation in these species may be associated with the relatively stable maritime temperature regime during the growing season along the Peninsula. In contrast to P(n), both species exhibited full acclimation of R(d), and rates of R(d) on a leaf-area basis were similar among treatments when measured at their respective daytime growth temperature. Our results suggest that in the absence of interspecific competition, continued warming along the Peninsula will lead to improved vegetative growth of these species due to (1) greater biomass allocation to leaf-area production (as opposed to improved rates of P(n) per leaf area) and (2) their ability to acclimate R(d), such that respiratory losses per leaf area do not increase under higher temperature regimes.     

Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats

The compounds resulting from the reaction of glucose with proteins (advanced glycation products) can be important markers of chronic diabetic complications. To test the possible diagnostic value of advanced glycation products containing the furoyl moiety, collagen samples from diabetic and healthy rats were analyzed by parent ion spectroscopy. In our study, we compared normal collagen, diabetic collagen and normal collagen incubated with different glucose concentrations and we employed different hydrolysis procedures (HCl and proteinase). Mass spectroscopic measurements performed on hydrolyzed samples showed that either different samples or different hydrolysis procedures produce a similar set of furoyl-containing compounds. 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) which has been reported to be one of the advanced glycation products, was never found in any of the samples examined. Hence neither FFI nor furoyl-containing molecules can be considered markers of advanced glycation processes.     

Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram

The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72h at 4°C. For AT the testicles were transported in normal saline in a container (17.9-21.5°C) where as for RT the testicles were transported in an ice-chest (4.9-6°C). The results of the current study revealed that intact acrosome was significantly higher (P<0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P>0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70-80%). The mean sperm motility for RT and AT at 0h was 82.50% and 75.00%, respectively and the corresponding values at 72h of preservation were 60.00% and 45.83%. The mean live sperm at 0h for RT and AT were 92.92% and 88.92%, respectively and the corresponding figures at 72h were 81.50% and 73.17%. The mean intact acrosome at 0h for RT and AT was 98.58% and 90.58%, respectively and at 72h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P<0.05) from 0h to 72h of preservation for both transportation temperatures. The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The major sperm abnormality for both RT and AT at each hour of preservation up to 72h was less than 0.5%. The study concluded that epididymides or testicles should be transported to the laboratory at RT (4.9-6°C) either in an ice-chest or portable refrigerator for their processing, evaluation and storage.     

Thermal denaturation studies of collagen by microthermal analysis and atomic force microscopy

The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques.     

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.     

Effect of elevated CO2 and high temperature on seed-set and grain quality of rice

Hybrid vigour may help overcome the negative effects of climate change in rice. A popular rice hybrid (IR75217H), a heat-tolerant check (N22), and a mega-variety (IR64) were tested for tolerance of seed-set and grain quality to high-temperature stress at anthesis at ambient and elevated [CO(2)]. Under an ambient air temperature of 29 °C (tissue temperature 28.3 °C), elevated [CO(2)] increased vegetative and reproductive growth, including seed yield in all three genotypes. Seed-set was reduced by high temperature in all three genotypes, with the hybrid and IR64 equally affected and twice as sensitive as the tolerant cultivar N22. No interaction occurred between temperature and [CO(2)] for seed-set. The hybrid had significantly more anthesed spikelets at all temperatures than IR64 and at 29 °C this resulted in a large yield advantage. At 35 °C (tissue temperature 32.9 °C) the hybrid had a higher seed yield than IR64 due to the higher spikelet number, but at 38 °C (tissue temperature 34-35 °C) there was no yield advantage. Grain gel consistency in the hybrid and IR64 was reduced by high temperatures only at elevated [CO(2)], while the percentage of broken grains increased from 10% at 29 °C to 35% at 38 °C in the hybrid. It is concluded that seed-set of hybrids is susceptible to short episodes of high temperature during anthesis, but that at intermediate tissue temperatures of 32.9 °C higher spikelet number (yield potential) of the hybrid can compensate to some extent. If the heat tolerance from N22 or other tolerant donors could be transferred into hybrids, yield could be maintained under the higher temperatures predicted with climate change.