Isolates of cell types from sorghum stems: Digestion,cell wall and anatomical characteristics

Cell types were separated from internode 5 of sorghum stems to study the interrelationship between digestion characteristics and cell wall composition. Isolates of epidermis (EPI), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) were freeze-dried and ground for analysis. The cell fractions were digested in rumen fluid for times between 0 and 96 h, and wall composition measured using detergent extraction procedures. In-vitro dry matter digestibility (g kg^?1 after 48 h) of cell fractions was in the order of PITH (849-906) > IVB (794-816) > SCL (692-701) > VBZ (641-679) > EPI (608-628). Total cell wall content (CWC), indigestible CWC, and lignin content followed the inverse order. Lignin concentration on a dry matter or cell wall basis was highly correlated with indigestible wall residue after 96 h. The proportion of cell wall digested after 96 h was higher for SCL and VBZ cells (61·8-68·2%) than for PITH cells (48·4-56·1 %), despite the former having lignin content three to five times higher than that of PITH cells. Clearly, there were differences between the cell types in wall composition or chemical linkages between wall components that lead to the observed differences in wall digestion.     

The thermomechanical properties of carrot cell wall material

The effect of temperature and moisture on the fabrication of pressed carrot cell wall specimens for Dynamic Mechanical Thermal Analysis was assessed. Results obtained from the water extractability of the material showed that more cell wall material became solubilised when moisture and temperature of the different treatments were increased. Chemical analysis revealed that this involved an increase in the water-soluble uronic acid components. Furthermore, more water-soluble neutral monosaccharides were observed, represented principally by galactose, rhamnose, arabinose and glucose. Pectic polysaccharides became more water soluble when isolated carrot cell wall was pressed at 100°C with a water content 800 g kg⁻¹ (wet weight basis). A molecular weight fraction centred at 100000 Da was observed in the severely pressed material (100°C, 800 g kg⁻¹ water) but was barely present in the mildly pressed (30°C, 500 g kg⁻¹ water) and unpressed specimens, consistent with depolymerisation and solubilisation. In contrast to the chemical modifications, the bending modulus, E′, of the pressed carrot cell wall material remained unchanged for the cell wall specimens moulded under different conditions, consistent with small changes in molecular weight. Pressed cell wall material was stiffer than pressed freeze-dried carrot which could be due to the plasticising role of the intracellular components. The stiffness of both cell wall and freeze-dried carrot specimens decreased with plasticisation by water in the range 10–500 g kg⁻¹. © 1998 Society of Chemical Industry.     

Determination of Chlorogenic Acids with Lactones in Roasted Coffee

An HPLC method has been developed which allowed the determination of mono- and dicaffeoylquinic acids (CQA and di-CQA), corresponding lactones (CQL) and feruloylquinic acids (FQA) in roasted coffee within one chromatographic run. The elution order was verified by isolation of the individual compounds by preparative HPLC, chromatography of the fractions on the analytical HPLC system, NMR spectroscopy and thermospray LC-MS. At least 15 additional minor compounds had the spectra of hydroxycinnamic acids, some of them only occurring in roasted coffee. Caffeoyltryptophan, which has been identified some time ago by another working group could also be separated with this system. The average contents in commercial roasted coffee samples (n=12) were: 3-CQA, 5·0 g kg⁻¹; 4-CQA, 6·2 g kg⁻¹; 5-CQA, 11·4 g kg⁻¹; 4-FQA, 0·7 g kg⁻¹; 5-FQA, 1·4 g kg⁻¹; 3-CQL, 2·1 g kg⁻¹; 4-CQL, 1·0 g kg⁻¹; 3,4-di-CQA, 0·7 g kg⁻¹; 3,5-di-CQA, 0·4 g kg⁻¹ and 4,5-di-CQA, 0·8 g kg⁻¹ dry matter. There were only small differences between the different brands. Two series of samples with different degrees of roast produced from the same green coffee have also been analysed. One series was steam treated before roasting (a process which is commercially used to improve digestibility). Only in the initial stages of roasting (light roast) could a difference in the contents of the acids between both series be observed. Keeping coffee brews at an elevated temperature (4 h at 80°C) reduced the amounts of CQL to 60% of the initial value. The contents of 3-CQA and 4-CQA increased, whilst that of 5-CQA decreased. The overall contents of CQA decreased.     

In vitro protein digestibility and mineral availability of green beans (Phaseolus vulgarisL) as influenced by variety and pod size

Three varieties of green beans (Cleo, Strike and Sentry) were harvested and sorted into four fractions according to pod size (diameter <7 mm; 7–8·5 mm; 8·6–10 mm and >10 mm). Ash content and dietary fibre increased significantly as pod size increased mainly in Cleo and Strike beans. Strike showed the highest fibre content (378·0 g kg⁻¹) but the lowest carbohydrate (364·6 g kg⁻¹) and ash (68·4 g kg⁻¹) values. Mean values for Fe and Mg content were higher in Cleo beans (70·9 and 27·1 mg kg⁻¹, respectively), Zn, Cu and Mg were higher in Strike beans (48·7 mg kg⁻¹, 22·4 mg kg⁻¹ and 3·15 g kg⁻¹, respectively) while Na and Ca values were maximum in Sentry (459·1 mg kg⁻¹ and 7·11 g kg⁻¹, respectively). Trypsin inhibitor was negatively related to in vitro protein digestibility but no relationship was found between this last parameter and phytic acid content. This antinutrient, together with dietary fibre, and a negative influence on in vitro mineral dialysability of green beans. © 1998 SCI     

Effects of conventional boiling on the polyphenols and cell walls of pears

Pears of the cultivar Gieser Wildeman were cooked for up to 24 h and changes in polyphenol and cell wall components were monitored. The main polyphenols were flavan‐3‐ols (epicatechin and its procyanidin oligomers), with an average degree of polymerisation of 6, and caffeoylquinic acid. Upon cooking, flavan‐3‐ols were retained in the pear tissue while the hydroxycinnamic acids were partially leached into the cooking water. After 1 h of cooking, 65% of the original flavan‐3‐ols and 40% of the original caffeoylquinic acid were still detectable in the pear tissue; the cooking water contained only 2% of the flavan‐3‐ols but 24% of the caffeoylquinic acid. Cell walls represented 23 g kg^?1 of the fresh pear and were composed of cellulose, pectins and xylans. The pectic fractions was degraded during cooking while xylans and cellulose were not affected. Copyright © 2004 Society of Chemical Industry     

Diferulate cross-links impede the enzymatic degradation of non-lignified maize walls

We assessed the effect of ferulate substitution and diferulate cross-linking of xylans on the degradation of cell walls by two fungal enzyme mixtures, one of which contained feruloyl esterase and high xylanase activities. Non-lignified cell suspensions of maize (Zea mays) were grown with 0 or 40 μM 2-aminoindan-2-phosphonic acid to produce walls with normal (17·2 mg g⁻¹) or reduced (5·1 mg g⁻¹) ferulate concentrations. Walls were incubated with mercaptoethanol to inhibit diferulate formation or with hydrogen peroxide to stimulate diferulate formation by wall bound peroxidases. Varying the ferulate substitution of xylans did not affect cell wall hydrolysis. In contrast, increasing ferulate dimerisation from 18 to 40% reduced carbohydrate release by 94–122 mg g⁻¹ after 3 h and by 0–48 mg g⁻¹ after 54 h of enzymatic hydrolysis. Diferulate cross-links impeded the release of xylans, cellulose and pectins from walls. These results provide compelling evidence that diferulate cross-links reduce the rate and, to a lesser degree, the extent of wall hydrolysis by fungal enzymes. Our results also suggest that enzyme mixtures containing high xylanase activity but not feruloyl esterase activity can partially overcome the inhibitory effects of diferulate cross-linking on wall hydrolysis. © 1998 SCI.     

Characterisation of peptides in silages made from perennial ryegrass with different silage additives

The effects of applying either formic acid (5.4 g kg⁻¹), a mixture of formic acid (2.7 g kg⁻¹) and formaldehyde (1.5 g kg⁻¹, 81 g kg⁻¹ herbage crude protein) or two concentrations of a cysteine peptidase inhibitor, cystamine (5 or 50 g kg⁻¹), to perennial ryegrass (Lolium perenne) on the nitrogen (N) distribution of the resulting silages were investigated, with emphasis on changes in concentration, composition and molecular weight of silage peptides. Herbage (156 g dry matter kg⁻¹ and 141 g water‐soluble carbohydrate kg⁻¹ dry matter) was ensiled in triplicate in laboratory silos for 100 days. Formic acid and the formic acid/formaldehyde mixture reduced soluble non‐protein N and ammonia N concentrations (P < 0.01); in addition, formic acid increased peptide N concentrations (P < 0.05). Cystamine at 50 g kg⁻¹ reduced ammonia N concentrations (P < 0.01) and increased peptide N concentrations (P < 0.05), but when applied at 5 g kg⁻¹ had little effect. Gel filtration of silage extracts on Sephadex G‐25 suggested that a small proportion (0.06–0.11 g kg⁻¹ peptide N) of silage peptides had a chain length of 7–9 amino acids, but remaining peptides were smaller with chain lengths of 2–6 amino acid residues. Amino acid analysis of silage peptides indicated that additive treatment had little effect on peptide amino acid composition but that peptides with a chain length of 7–9 amino acids contained lower proportions of isoleucine and arginine. © 2000 Society of Chemical Industry     

Proximate Composition and Seed Lipid Components of Sorghum Cultivars from Argentina

Seeds of 11 sorghum cultivars ( Sorghum bicolor ) from Argentina were analysed for proximate composition, fatty acids and sterols. Oil, protein, carbohydrate and ash contents varied between 41 and 66 g kg⁻¹, 111 and 156 g kg⁻¹, 670 and 730 g kg⁻¹ and 13·8 and 20·6 g kg⁻¹ of dry matter, respectively. Fatty acid profiles revealed that the major acids were palmitic (15·1–24·8%), oleic (29·9–41·8%) and linoleic (35·9–51·3%). Unsaponifiable matter was examined for sterols. Sitosterol was the prominent component in all cultivars (43·8–57·9%), followed by campesterol (18·7–29·1%) and stigmasterol (12·4–20·5%).     

Effects of different additives on the cell wall and mineral fractions of artichoke (Cynara scolymusL) and orange (Citrus aurantiumL) by-product silage

The quality of the cell wall fraction content of two silage by-products was assessed together with their different mineral composition. Various treatments to test possible improvements in the ensilage of canning industry artichoke and orange by-products were compared with the addition of different additives. Three treatments were assessed: sodium chloride (25 g kg⁻¹), dehydrated beet pulp (62·5 g kg⁻¹), formic acid at 4% in doses of 62·5 ml kg⁻¹. A fourth batch acted as a control group. Samples were analysed for their nutritive characteristics. After a 100-day ensilage period the results showed that the use of the additives in the given doses did not significantly improve the quality of the silage, although the composition of the sodium chloride batch appeared to be better. © 1998 SCI.     

Characterization of p‐coumarate accumulation,p‐coumaroyl transferase,and cell wall changes during the development of corn stems

BACKGROUND: Developmental changes occur in corn (Zea mays L.) stems from cell initiation to fully mature cell types. During cell wall maturation the lignin is acylated with p‐coumarates (pCA). This work describes characterization studies of the p‐coumaroylation process in relation to corn stem development. RESULTS: Corn plants from three locations were harvested and tissues were analyzed from all nodes and even‐numbered internodes above soil line. Changes in carbohydrates reflect a shift to lignification at the expense of structural polysaccharide synthesis. Accumulation of pCA paralleled the incorporation of lignin while ferulate (FA) remained relatively constant as a proportion of the cell wall (5–7 g kg^?1 CW). The p‐coumaroyl transferase (pCAT), which is responsible for attaching pCA to lignin monomers, displayed maximum levels of activity in the middle region of the stem (internodes 10–12, 2–3 nmol L^?1 min^?1 mg^?1). The syringyl content as a proportion of the total lignin did not change significantly with cell wall maturation although there was a trend towards increased amounts of syringyl units in the more mature cell walls. CONCLUSIONS: Incorporation of pCA into corn cell walls not only mirrored lignification but the pCAT activity as well. Levels of pCAT activity may be an indicator of rapid lignification specifically for syringyl type lignin. Copyright © 2008 Society of Chemical Industry     

Lipid composition of Bulgarian chokeberry,black currant and rose hip seed oils

The lipid composition of chokeberry, black currant and rose hip seeds was investigated. The seeds contain 19.3 g kg⁻¹, 22.0 g kg⁻¹ and 8.2 g kg⁻¹ glyceride oil respectively. The content of phospholipids, mainly phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine, was 2.8 g kg⁻¹, 1.3 g kg⁻¹ and 1.4 g kg⁻¹, respectively. The total amounts of sterols were 1.2 g kg⁻¹, 1.4 g kg⁻¹ and 0.4 g kg⁻¹. The main component was β-sitosterol, followed by campesterol and Δ⁵ -avenasterol. In the tocopherol fraction (55.5 mg kg⁻¹ in chokeberry oil, 249.6 mg kg⁻¹ in black currant oil and 89.4 mg kg⁻¹ in rose hip oil), α-tocopherol predominated in chokeberry oil (70.6 mg kg⁻¹). γ-Tocopherol was the main component in black currant oil (55.4 mg kg⁻¹) and rose hip oil (71.0 mg kg⁻¹). The fatty acid composition of triacylglycerols, individual phospholipids and sterol esters was also identified. In the phospholipids and sterol esters, the more saturated fatty acids, mainly palmitic, stearic, and long chain fatty acids predominated. © 1999 Society of Chemical Industry     

Aleurone cells are the primary contributor to arabinoxylan oligosaccharide production from wheat bran after treatment with cell wall-degrading enzymes

Arabinoxylans and in particular arabinoxylan oligosaccharides (AXOS) from wheat are recognised for their prebiotic potential. A high-yield, non-chemical production of AXOS is therefore of interest when producing functional foods. This study investigated the enzymatic production of AXOS from wheat bran with the aim of establishing the main fraction contributing to production of AXOS. Fractions of wheat bran, outer pericarp and aleurone with two different purities were treated with the cell wall-degrading enzymes: xylanase, cellulase and β-glucanase. The yield of solubilised arabinoxylans upon treatment was greatest in the most pure aleurone fraction (164 g kg⁻¹) and lowest in the outer pericarp fraction (15 g kg⁻¹). The yield was mainly recovered as AXOS rather than soluble arabinoxylans and was negatively related to the arabinose/xylose ratio found in the raw material. In conclusion, wheat aleurone cell walls are the main contributor to the production of AXOS from wheat bran and this seems to depend on the A/X ratio of the raw material.     

Selenium analysis of selected Egyptian foods and estimated daily intakes among a population group

The selenium (Se) content of 67 local foods had been determined using electrothermal (ETAAS) and hydride generation (HGAAS) atomic absorption spectrometry. The richest sources of Se were chicken (323±35 μg kg⁻¹); eggs (166±19.4 μg kg⁻¹) and fish (306±5.2 μg kg⁻¹). Vegetables and fruits contained trace amounts of Se (1–33 μg kg⁻¹). Wide variations in Se content were reported between various bread types and values ranged from 14 (home-made bread) to 152 (flattened bread) μg kg⁻¹, respectively. The estimated adult Se intake was 49 μg kg⁻¹; bread being the major contributor (63.6%). ©     

Dietary fibre content of thirteen apple cultivars

Fibre composition of the following 13 apple cultivars was studied: ‘Cortland’, ‘Empire’, ‘Fuji’, ‘Golden Delicious’, ‘Gala’, ‘Granny Smith’, ‘Jonagold’, ‘Mutsu’, ‘McIntosh’, ‘Delicious’, ‘Rome’, ‘Stayman’ and ‘York’. Fruit samples from each of these cultivars were analysed for non-starch cell wall materials (NSCWM) and non-starch polysaccharides (NSP). NSCWM was further fractionated into soluble and insoluble fibre fractions. Both NSCWM and NSP content were found to be significantly influenced by cultivar. NSCWM content ranged from 19·1 g kg⁻¹ apple flesh in ‘Fuji’ to 36·2 g kg⁻¹ in ‘York’. Mean(±SD) NSCWM content of all the cultivars was 23·1±4·5 g kg⁻¹. NSP content of apple flesh ranged from 13·8 g kg⁻¹ in ‘McIntosh’ to 28·7 g kg⁻¹ in ‘York’ with the overall mean for all cultivars being 17·9±4·2 g kg⁻¹. Relative amount of monosaccharides found in the hydrolysates of apple fibre also varied among cultivars. The greatest difference was observed in galactose content. ©1997 SCI     

Effect of fermentation and bacterial inoculation on lucerne cell walls

Changes were found in the cell wall composition of lucerne after ensiling at three different dry matter (DM) contents. The amount of protein associated with cell walls was reduced during ensiling, regardless of inoculation level, by 46–68%, whereas protein associated with the lignin residue was reduced to a lesser extent (< 40%). The effect ofensiling on individual sugars of the cell wall varied. Uronics of the cell wall were decreased by 12% with wilted silages (290 g DM kg^–1) but were unchanged in a higher dry matter silage (401 g DM kg^–1) and silages with a limited pH change during ensilage. The arabinose and galactose contents, as a fraction of cell walls, decreased (15–24%), increasing glucose and xylose contents proportionally. Inoculation decreased arabinose and galactose contents early in the fermentation when the pH decline was enhanced but final values were not significantly different (P > 0.32). Removal of the sugars from the cell wall appears to be related to pH because silages with little pH change had no change in the cell wall sugars and inoculation reduced the cell wall sugars only after silage pH declined.     

Effect of extrusion on nutritional quality of maize and Lima bean flour blends

Maize flour (Zea mays) (M), Lima bean flour (Phaseolus lunatus) (B) and blends of these in proportions of 75M/25B, 50M/50B and 25M/75B (w/w) were extruded and their nutritional quality evaluated. Extrusion was done with a single screw extruder at 160 °C, 100 rpm and 15.5% moisture. In vitro protein digestibility (87%) was higher in the extruded products. Available lysine and resistant starch were highest in 50M/50B raw flour (59.5 g kg⁻¹ protein, 67.9 g kg⁻¹, respectively) but decreased after extrusion (45.5 g kg⁻¹ protein, 16.6 g kg⁻¹, respectively). The same treatment had the lowest available starch (561.6 g kg⁻¹ flour, 507.9 g kg⁻¹ extrudate). Total dietary fiber in the 50M/50B raw flour blend was 144 g kg⁻¹ versus 176 g kg⁻¹ in its extrudate. This was most noticeable for soluble dietary fiber, which increased from 10.6 g kg⁻¹ to 79.4 g kg⁻¹ after processing. Extrusion of blends is feasible up to a 50% bean inclusion level, which improves the nutritional value of the expanded product.Copyright © 2006 Society of Chemical Industry     

Fermentation characteristics of polysaccharide fractions extracted from the cell walls of maize endosperm

Cell walls were extracted from maize endosperm and separated into different polysaccharide fractions by sequential extraction with solutions of saturated Ba(OH)₂, demineralised water and 1 and 4 M KOH. Solubilised polysaccharides were collected after each extraction. Residues were collected following the extractions with demineralised water and 1 and 4 M KOH. The original cell wall (CW) material, extracts and residues were analysed for their fermentation characteristics using an in vitro cumulative gas production technique. The rate of fermentation of the alkali‐treated residues was faster than that of the original CW material, except for the 4 M KOH residue, which had a similar rate of degradation to the original CW material. The polysaccharides solubilised from the cell wall (extracts) were all rapidly fermented, more rapidly than both CW and residues. A division of the gas production profile into two phases using curve fitting was in good agreement with a division of the cell wall fermentation into the fermentation of arabinoxylans and cellulose. Therefore the likelihood of preferential degradation of arabinoxylans from the maize cell wall was discussed. The volatile fatty acid production pattern was fairly well explained by the fermentation rate and composition of the substrates. It was concluded that breaking the interactions of polysaccharides in the maize cell wall by mild alkali extraction increases the fermentability of maize cell walls in the gastrointestinal tract of farm animals. Contrarily, more severe alkali extractions will reduce the fermentability of maize cell walls. © 2002 Society of Chemical Industry     

Preparation and composition of mesophyll,epidermis and fibre cell walls from leaves of perennial ryegrass (lolium perenne) and italian ryegrass (lolium multiflorum)

Cells of mesophyll, epidermis and residual fibrous material were obtained from leaves of Italian and perennial ryegrass harvested at different stages of maturity by mechanical disruption of leaf tissue. Mesophyll cells were selectively removed by filtration through 0.045 mm nylon mesh and remaining non-mesophyll cells centrifuged in metrizamide solutions (56–58% wt to vol.) of known density (1.308–1.329 g cm³ at 5°C) to obtain a pure epidermis cell fraction and a residual fibre fraction. Whole mesophyll cells contributed 63–72%, epidermis 12–15% and the fibre fraction 15–24% to the total leaf dry matter. Fibre values were higher in late-cut samples. Cell walls were prepared from mesophyll and epidermis cells by disruption and washing to remove cell contents. Fibre cells were judged free of cell contents and received no further treatment. Examination of cell wall preparations by light and electron microscopy showed that both mesophyll and epidermis preparations were essentially free from contaminating material. Mesophyll cell walls were uniformly thin (200 nm) while those of epidermis ranged from 2000–3000 nm at the outer face, thinning to 300 nm or less at the inner surface. An electron-light layer (cuticle) of approximately 200 nm thickness was present covering the outer face of the epidermis. The fibre fraction largely consisted of sclerenchyma, but contained, in addition, other vascular cells, detached annular rings and heavily silicified leaf hairs. Analysis of cell walls accounted for 85–90% of dry matter. Cellulose was the major component of all cell walls examined (approximately 40% of dry matter) with xylose residues accounting for a further 11% of mesophyll, 13.5–17.5% of epidermis and 21–25% of fibre cell walls. Arabinose was low in fibre cells but was present in much higher proportions in mesophyll and epidermis walls. The ratio of arabinose to xylose was approximately 1:1.5 for mesophyll, 1:2.5 for epidermis and 1:7.0 for the fibre fraction. The molar ratio acetyl to xylose remained fairly constant at 1:4 regardless of the grass, cell type or maturity of the sample. The uronic acid content of epidermis was higher than that in other cell types and showed an increase with increasing maturity of the grass, reaching over 9% in late-cut samples. Total phenolic material represented 2–3% of mesophyll and epidermis cell walls and 6% of fibre walls. Ferulic acid alone was released from the primary cell walls by saponification and p-coumaric and ferulic acids from the secondary-thickened fibre walls. Crude protein values (NX6.25) were high in mesophyll cell wall preparations and low in epidermis and fibre cell walls. Amino acid patterns were similar for both grasses and cell types but hydroxyproline was found in greater amounts in fibre cell walls than in either epidermis or mesophyll.     

Fatty Acid Composition of ‘Cerrado’ Seed Oils

The seeds of 28 species from ‘cerrado’, a typical savanna ecosystem of Brazil, were analysed for total lipid contents and fatty acid distribution. The seeds of 10 species presented contents above 150 g kg⁻¹, the highest yield reaching 335 g kg⁻¹. Distribution of fatty acids based on polyunsaturated compounds seems to be rare in seed oils from ‘cerrado’: only three seed oils were found to be based on linoleic acid and none on linolenic acid. Eight seed oils, four of them Fabales, presented palmitic acid as a dominant constituent. Half of the species presented oleic acid based seed oils. Two species stand out for unusual fatty acid distribution: Qualea grandiflora (Vochysiaceae) with 171 g kg⁻¹ of seed oil presenting 723 g kg⁻¹ of lauric acid and Serjania erecta (Sapindaceae) with 256 g kg⁻¹ of seed oil presenting 623 g kg⁻¹ of eicosenoic acid.     

Toxicological evaluation of acetylated faba bean globulin

Acetylated faba bean globulin (acVFG, acetylation degree 97%) was evaluated toxicologically in a subchronic feeding study and a female fertility test. Groups of male and female rats received acVFG in their diet at levels of 20, 40 and 80 g kg⁻¹ for 3 months. Additional groups were fed a commercial diet or given food containing 38 or 75 g kg⁻¹ casein. The subchronic toxicity study revealed growth retardation in both sexes at the 80 g kg⁻¹ acVFG level. The food consumption was reduced in males at 40 and 80 g kg⁻¹ acVFG and in females at 80 g kg⁻¹ acVFG. Histopathological alterations in the ovaries were observed at dietary levels of 40 and 80 g kg⁻¹ acVFG. Histopathological changes and decreases in the absolute and relative weight of the uterus were found in all groups fed acVFG. Therefore a no‐observed‐effect level (NOEL) was not derivable. Feeding females 80 g kg⁻¹ acVFG resulted in diminished pre‐implantation losses but increased post‐implantation losses in the fertility study. © 2000 Society of Chemical Industry