Biotechnology and Plant Disease Control-Role of RNA Interference

Development of crop varieties which are resistant against many economically important diseases is a major challenge for plant biotechnologists worldwide. Although much progress in this area has been achieved through classical genetic approaches, this goal can be achieved in a more selective and robust manner with the success of genetic engineering techniques. In this regard, RNA interference (RNAi) has emerged as a powerful modality for battling some of the most notoriously challenging diseases caused by viruses, fungi and bacteria. RNAi is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid (dsRNA) inhibits the expression of genes with complementary nucleotide sequences. The application of tissue-specific or inducible gene silencing in combination with the use of appropriate promoters to silence several genes simultaneously will result in protection of crops against destructive pathogens. RNAi application has resulted in successful control of many economically important diseases in plants.

Empty nose syndrome pathogenesis and cell-based biotechnology products as a new option for treatment

Empty nose syndrome (ENS) is a rare complication that develops after partial orcomplete turbinectomy. The main feature of ENS is paradoxical nasal obstructionfeeling despite objectively wide nasal airway. ENS pathogenesis is multifactorialand includes changes in laminar physiological airflow, disruption of mucosafunctions and deficient neural sensation. This leads to the development of ENSsymptomatology such as dyspnea, nasal dryness, nasal burning, nasalobstruction, feeling of suffocation and even comorbid psychiatric disorders thatsignificantly impairs life quality. Specific effective treatment of ENS does not existup to date. In this review we outline existing biomaterial for surgical reconstitutionof nasal anatomy and discuss the perspective of stem cell-based technologiesin ENS management. The main focus is directed to justification ofrationality application of adult mesenchymal stem cells (MSCs) from differenttissues origin and neural crest-derived stem cells (NCSCs) based on their intrinsicbiological properties. MSCs transplantation may stimulate mucosa tissueregeneration via trophic factors secretion, direct transdifferentiation into epithelialcells and pronounced immunosuppressive effect. From the other hand, NCSCsbased on their high neuroprotective properties may reconstitute nerve structureand functioning leading to normal sensation in ENS patients. We postulate thatapplication of cell-based and tissue-engineered products can help to significantlyimprove ENS symptomatology only as complex approach aimed at reconstitutionof nasal anatomy, recovery the nasal mucosa functionality and neural tissuesensation.

Neoantigen cancer vaccines:a new star on the horizon

Immunotherapy represents a promising strategy for cancer treatment that utilizes immune cells or drugs to activate the patient's own immune system and eliminate cancer cells.One of the most exciting advances within this field is the targeting of neoantigens,which are peptides derived from non-synonymous somatic mutations that are found exclusively within cancer cells and absent in normal cells.Although neoantigen-based therapeutic vaccines have not received approval for standard cancer treatment,early clinical trials have yielded encouraging outcomes as standalone monotherapy or when combined with checkpoint inhibitors.Progress made in high-throughput sequencing and bioinformatics have greatly facilitated the precise and efficient identification of neoantigens.Consequently,personalized neoantigen-based vaccines tailored to each patient have been developed that are capable of eliciting a robust and long-lasting immune response which effectively eliminates tumors and prevents recurrences.This review provides a concise overview consolidating the latest clinical advances in neoantigen-based therapeutic vaccines,and also discusses challenges and future perspectives for this innovative approach,particularly emphasizing the potential of neoantigen-based therapeutic vaccines to enhance clinical efficacy against advanced solid tumors.

Differences in leaf cuticular wax induced by whole-genome duplication in autotetraploid sour jujube

Drought-resistant plants exhibit strong water retention capability.In this regard,the autotetraploid sour jujube leaves exhibit better water retention than diploid leaves.Morphological comparisons and physiological comparisons of diploid and autotetraploid leaves showed that the autotetraploid leaves had thicker leaf cuticles and more leaf wax accumulation than the diploid leaves,which could reduce cuticle permeability and improve the drought tolerance of leaves.In this study,the cuticular wax crystalloids on the adaxial and abaxial sides of young and mature jujube leaves were observed in the two ploidy types,and unique cuticular wax crystalloids covering a large area of the cuticle on autotetraploid sour jujube leaves may provide an advantage in reducing leaf non-stomata transpiration and improving plant drought tolerance.Based on the transcriptome,115 differentially expressed genes between diploids and autotetraploids were further analyzed and found to be involved in the accumulation of cuticular wax components,including terpenoids,fatty acids,and lipids,as well as ABC transporter and wax biosynthetic process.Finally,14 genes differentially expressed between glossy autotetraploid leaves and nonglossy diploid leaves,such as LOC107414787,LOC107411574 and LOC107413721,were screened as candidate genes by qRT-PCR analysis.This findings provided insights into how polyploidization improved drought tolerance.

Potential of photodynamic therapy in the management of infectious oral diseases

Photodynamic therapy(PDT)can take place in the presence of three elements:Light with an appropriate wavelength;a photosensitizer;and the presence of oxygen.This type of treatment is very effective overall against bacterial,viral and mycotic cells.In the last 10 years many papers have been published on PDT with different types of photosensitizers(e.g.,methylene blue,toluidine blue,indocyanine green,curcumin-based photosensitizers),different wavelengths(e.g.,460 nm,630 nm,660 nm,810 nm)and various parameters(e.g.,power of the light,time of illumination,number of sessions).In the scientific literature all types of PDT seem very effective,even if it is difficult to find a standard protocol for each oral pathology.PDT could be an interesting way to treat some dangerous oral infections refractory to common pharmacological therapies,such as candidiasis from multidrug-resistant Candida spp.

Nutritional and Clinical Profiles of Elderly People with Diabetes and Hypertension at Conakry University Hospitals

Introduction: In Guinea, there is few data on dietary practices socio-economic and clinical characteristics of elderly diabetics and hypertensives. Methods: This was a descriptive cross-sectional study of people aged 60 years and over, seen in consultation or hospitalised in Conakry University hospitals during the period from 5 September to 15 December 2023 inclusively. Results: Among the 320 subjects included in the study, there were more men (50.6%). The average age was 67 ± 7.69 years;53.43% had hypertension and 46.57% had diabetes. Sex was significantly associated with diabetes (p = 0.035). Women in the study were more affected by diabetes (55.7%), 64.4% of diabetic subjects were married and more than 64% of them had dependent children. 26.2% of diabetics were not employed;85.9% had an income;34.9% had an income of less than one million Guinean francs per month and 74.5% of them had 3 meals a day. Physical activity, 24-hour recall for lunch (p Conclusion: Effective management of diabetes and hypertension in the elderly should necessarily involve nutrition education in hospitals.

Production of Yam Tubers Using Seed Tubers from Vitroplants Regenerated from Aerial Stems in the Yam Species Dioscorea alata (L.) and Dioscorea cayenensis-rotundata (L. & P.) in Côte d’Ivoire

The aim of this study is to produce large quantities of yam tubers in the field from seed tubers derived from vitroplants regenerated from aerial stem cuttings and farmers’ tuber seeds. Seed tubers from ten yam cultivars, including five from Dioscorea alata (cDa083, cDa053, cDa115, cDa150 and cDa266) and five from Dioscorea cayenensis-rotundata (cDr015, cDr027, cDr150, cDr206 and cDr148) with distinct agro-morphological characteristics, were used as material. A completely randomized block design with 3 replications was used. In each block, all cultivars and seed types were represented. When put into cultivation, the tubers produced by the vitroplants all germinated and gave 100% healthy plants, compared with 86% to 100% healthy plants for the seed tubers produced by the farmers. Yields per hectare ranged from 10 to 25 tonnes for seed tubers produced by in vitro plants, and from 4 to 9 tons for seed tubers produced by farmers. Seed tubers from vitroplants can therefore be recommended to farmers as a solution to the problem of seed tuber unavailability.

Soil Physico-Chemical Properties and Different Altitudes Affect Arbuscular Mycorrhizal Fungi Abundance and Colonization in Cacao Plantations of Cameroon

This study aims to investigate the abundance of AMF according to soil properties and altitudes in different cacao plantations of Cameroon. Physico-chemical analyses were made on soil samples collected from three agro-ecological zones. Soil samples were also used to evaluate directly the AMF abundance following the various altitudes and after trapping by sorghum plant. The results showed that soil properties, AMF spore abundances and colonization fluctuated significantly at different altitudes. The most represented texture was sandy loam. The bimodal zone presented a homogeneous texture (sandy loam) in all its localities. Cacao soil chemical characteristics showed that, the highest nitrogen rate (0.47%;p 0.05, Scott-Knott test) was recorded at Melong in a monomodal zone while Tonga in the Western highlands displayed the lowest rate (0.13%). Soil P concentration was significantly high in monomodal zones (Mbanga and Melong). Soil pH level indicated that the soil from Tonga in the Western highlands was neutral (pH = 6.67), and soils of other localities under study were acidic with the lowest (4.75) pH level recorded at Melong in a monomodal zone. In soil samples, the highest spore density (1.03 spores/g soil) was observed at Ntui in Bimodal zone, while the lowest spore density (0.26 spores/g soil) was observed at Bafang in the Western highlands. Root colonization showed that the sample from Bokito in a bimodal zone displayed the best frequency of mycorrhization (86.11%) while the sample from Bafang in the Western highlands recorded the lowest (27.11%). The PCA analysis highlighted that available phosphorus, pH and altitude all strongly correlated with AMF root colonization ability and can be used as a predictor of AMF colonization ability in cacao rhizosphere.

Profiling of the Retrospective Epidemiology of the Human Papillomavirus and Related Cancers in the United Arab Emirates Population

The human papillomavirus (HPV) is a leading cause of infectious cancers, leading to a growing global interest on the profiling of HPV-related cancers. Therefore, the aim of the study was to determine the retrospective epidemiological profile of HPV-related cancer in the United Arab Emirates (UAE). The incidence profiling of HPV-related cancers in males and females of UAE of all ages ranged from a minimum of 0.6% to a maximum of 4.5% for testes cancer and colon cancer respectively in males;whilst in females it ranged from a minimum of 1.0% and a maximum of 34% for Hodgkin lymphoma, kidney, bladder, liver and pancreas cancer and breast cancer, respectively. The incidence profiling of HPV-related cancers in males and females of ages 15 - 44 years old ranged from a minimum of 0.1% to a maximum of 11.2% for pancreas cancer, and colon and rectum cancer respectively in males, whilst in females it ranged from a minimum of 0.1% and a maximum of 18% for pancreas and breast cancer, respectively. The mortality profiling of HPV-related cancers in males and females of all ages in the UAE ranged from a minimum of 0.1% for Hodgkin lymphoma and testicular cancer to a maximum of 4.5% for colon and rectum cancer respectively in males, whilst in females it ranged from a minimum of 0.2% and a maximum of 4.5% for pancreas and breast cancer, respectively. The mortality profiling of HPV-related cancers mortality in males and females of ages 15 - 44 years old in the UAE ranged from a minimum of 0.2% to a maximum of 4.3% for testicular cancer and colon cancer respectively in males, whilst in females it ranged from a minimum of 0.2% and a maximum of 7.2% for bladder and breast cancer respectively. In conclusion, the profiling of HPV-related cancers in UAE for both males and females of all ages is similar to that reported for other parts of the world.

Safety and in Vivo Anti-Inflammatory Activity of Ethanolic Extract of Ficus umbellata (Vahl.) Leaves

Toxicity is the totality of adverse effects, which can be functional and morphological lesions in a living organism, caused by a substance introduced in relatively high single doses or in small, repeated doses. The aim of this study was to assess the OECD-recommended acute oral toxicity and anti-inflammatory activity of ethanolic extract of Ficus umbellata leaves. Animals were given a single oral dose of 1000, 3000 and 5000 mg/Kg body weight (BW) of the extract. For the anti-inflammatory activity test, rats were given the ethanolic extract of F. umbellata leaves at doses of 100, 300 and 500 mg/Kg or aspirin® at a concentration of 100 mg/Kg PC orally, one hour before injection of 0.05 ml of 1% formalin under the plantar fascia of the rat’s right hind paw. Paw volume measurements were taken one, two and three hours after formalin injection, using an electronic caliper. After 14 days of observation, no deaths were observed in treated rats. The LD50 of ethanolic extract of Ficus umbellata leaf powder is greater than 5000 mg/Kg body weight. This extract has no significant effects on hematological parameters and on the main markers of nephrotoxicity and hepatotoxicity for a single dose of less than 5000 mg/Kg PC. It reduces formalin-induced edema. Evaluation of the percentage inhibition showed that the extract had greater anti-inflammatory activity at 3 hours after the start of the experiment. However, better inhibition of inflammatory oedema of the paw of rats treated with 500 mg/Kg was observed at 5 hours after the start of the experiment, with a percentage inhibition of 69.23 ± 1.02, compared with the reference group treated with aspirin® 100 mg/Kg, which showed an inhibition of 63.50 ± 0.98. These results show that F. umbellata leaves possess anti-inflammatory activity, which would justify their use in traditional African medicine to prevent or treat inflammation.

Involvement of haptoglobin in disease development

Haptoglobin(HP)is a liver glycoprotein that is actively synthesized during in-flammatory and hemolytic processes.It also has pro-oxidant and proinflam-matory properties,which are a function of its genotype.The genetic polymorp-hism of the chains leads to synthesis of three phenotypes/proteins,which are related to the number and type of chains and their molecular weight,namely HP1-1,HP1-2 and HP2-2.Patients with HP2-2 have more vascular complications,while those with HP1-1 have fewer.HP is involved in the worsening of diseases,such as HP2-2 in aggravation of vaso-occlusive crises in sickle cell disease,and worsening of the pathophysiology of other diseases.In contrast,HP1-1 confers better protection against diseases.All of this suggests that further studies should be conducted,including experimental and analytical studies focused on de-monstrating the influence of different HP genotypes on individual clinical and hematological data.This would help in understanding the role played by this genetic polymorphism in the pathophysiology of diseases.

牛分离精子对体外受精的影响

本研究的目的是测定流动细胞分离仪分离的精子对牛卵母细胞体外受精和胚胎发育的影响。共使用了4273个牛的卵母细胞和3种类型的精子(分离、染色未分离和未染色未分离)进行体外受精试验。这些精子分别来自于3头公牛,每头公牛重复试验3～5次。数据使用ANOVA程序进行统计分析。结果显示,用3种类型精子受精后的卵母细胞囊胚率无显著差异,分别为20.4%,22.62%和22.14%,但用分离精子受精后的卵裂率显著低于未分离的精子(53.66%比71.93%,P<0.05)。所测试的3头公牛的卵裂率和囊胚率在不同公牛之间有所差异,H008号公牛所产生的囊胚明显低于H010号公牛组(分别为17.4%比25.1%,P<0.05)。结果证明分离过程或染色并没有影响胚胎发育,流动细胞分离仪可以有效地用于牛胚胎的体外生产。此外,分离、染色未分离和未分离精子的受精和胚胎发育率,在不同公牛之间有差异的这一发现表明,选择能产生高质量精子的公牛精液进行精子分离,可以提高体外受精的效率。

通过体外诱导突变及筛选来改良果树作物特性的研究进展(英文)

突变会导致栽培作物一个或多个特殊性状的改变,以突变为基础的植物品质的改良,会使果树业的发展获得极大的促进。传统诱导突变的方法应用于果树作物,常受限于外植体来源,并且耗费大量的时间及场地。体细胞变异结合物理化学诱变法可被视作改良果树作物的一种手段,并且在一些果树作物中获得了成功。这使我们有理由认为体外诱导变异是改良果树作物的品质及其抗逆性的一种很有效的潜在途径。本文讨论了大量的与果树培育有关的突变诱导、变异体筛选、大田评估方法等综合使用的实例及研究进展。

Effect of Nitrogen Management on Yield and Water Use Efficiency of Rainfed Wheat and Maize in Northwest China

A field experiment with four treatments and four replicates in a randomized complete block design was conducted at the Changwu Experimental Station in Changwu County, Shaanxi Province, of Northwest China from 1998 to 2002. The local cropping sequence of wheat, wheat-beans, maize, and wheat over the 4-year period was adopted. A micro-plot study using ^15N-lahelled fertilizer was carried out to determine the fate of applied N fertilizer in the first year. When N fertilizer was applied wheat （years 1, 2 and 4） and maize （year 3） grain yield increased significantly （P 〈 0.05） （〉 30%）, with no significant yield differences in normal rainfall years （Years 1, 2 and 3） for N application at the commonly application rate and at 2/3 of this rate. Grain yield of wheat varied greatly between years, mainly due to variation in annual rainfall. Results of ^15N studies on wheat showed that plants recovered 36.6%-38.4% of the N applied, the N remained in soll （0-40 cm） ranged from 29.2% to 33.6%, and unaccounted-for N was 29.5%-34.2%. The following crop （wheat） recovered 2.1%- 2.8% of the residual N from N applied to the previous wheat crop with recovery generally decreasing in the subsequent three crops （beans, maize and wheat）.

Hyperglycemia in acute ischemic stroke: physiopathological and therapeutic complexity

Diabetes mellitus and associated chronic hyperglycemia enhance the risk of acute ischemic stroke and lead to worsened clinical outcome and increased mortality. However, post-stroke hyperglycemia is also present in a number of non-diabetic patients after acute ischemic stroke, presumably as a stress response. The aim of this review is to summarize the main effects of hyperglycemia when associated to ischemic injury in acute stroke patients, highlighting the clinical and neurological outcomes in these conditions and after the administration of the currently approved pharmacological treatment, i.e. insulin. The disappointing results of the clinical trials on insulin(including the hypoglycemic events) demand a change of strategy based on more focused therapies. Starting from the comprehensive evaluation of the physiopathological alterations occurring in the ischemic brain during hyperglycemic conditions, the effects of various classes of glucose-lowering drugs are reviewed, such as glucose-like peptide-1 receptor agonists, DPP-4 inhibitors and sodium glucose cotransporter 2 inhibitors, in the perspective of overcoming the up-to-date limitations and of evaluating the effectiveness of new potential therapeutic strategies.

Fungal inoculation induces agarwood in young Aquilaria malaccensis trees in the nursery

Fungi are often used to induce agarwood inAquilariatrees. This study was conducted to evaluate the effect of several fungi on agarwood formation over time in youngAquilaria malaccensis （Lam.） trees. Typical changes in the length and light intensity of the resulting discoloration were observed after three and six month periods following inoculation. Wood samples were observed microscopically and classified into several light intensity groups. The discoloration length was meas-ured longitudinally. The duration after inoculation affected the mean of discoloration length： the 6-month old sample （1.70 cm） had a wider discoloration zone when compared to the 3-month old sample （1.17 cm）. When measuring the discoloration intensity, a positive relationship with time was perceived. Digital images, captured using a camera-equipped microscope, revealed that wood samples collected after six months appeared to be 1.8-times darker than after three months. We concluded that time, not the species of any of the tested fungi, had significant effect on discoloration length and intensity. Gas chromatography/mass spec-trometry （GCMS） analysis of the 6-month old sample yielded some important agarwood compounds such as benzylacetone, anisylacetone, guaiene and palustrol. This demonstrates that the tested fungi have the ability to induce agarwood formation in nurseryA. malaccensis trees.

Analysis of genetic relationship in mutant silkworm strains of Bom-byx mori using inter simple sequence repeat (ISSR) markers

Amplified inter simple sequence repeats （ISSR） markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat （SSR） motifs were used in this study. A total of 113 markers were produced among 20 mutant strains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was （1.7080 ± 0.4567）, effective alleles （1.5194 ± 0.3950） and genetic diversity （Ht） （0.2901 ± 0.0415）. The dendrogram produced using the unweighted pair group method with arithmetic means （UPGMA） and cluster analysis made using Nei＇s genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm strains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm strains maintained at the germplasm center.

小麦秸秆发酵产生的真菌Pleurotus ostreatus IBL-02锰过氧化物酶的溶胶-凝胶固定化及其催化污水脱色(英文)

Solid state bio-processing of wheat straw was carried out through an indigenous fungal strain Pleurotus ostreatus IBL-02 under pre-optimized fermentation conditions. The maximum activity, 692±12 U/mL, of the industrially important manganese peroxidase (MnP) enzyme was recorded after five days of still culture incubation. The crude MnP was 2.1-fold purified with a specific activity of 860 U/mg after purification on a Sephadex-G-100 gel column. On native and SDS-PAGE electrophoresis gels, the purified MnP fraction was a single homogenous band of 45 kDa. An active fraction of MnP was immobilized using hydrophobic sol-gel entrapment comprising tetramethoxysilane (T) and propyltrimethoxysilane (P) at different T:P molar ratios. Characterization revealed that after 24 h incubation at varying pH and temperatures, the MnP fraction immobilized at a T:P ratio of 1:2 in the sol-gel retained 82% and 75% of its original activity at pH4 and 70 ℃, respectively. The optimally active fraction at a 1:2 T:P ratio was tested against MnSO4 as a substrate to determine the kinetic catalytic constants KM and Vmax . To explore the industrial applicability of P. ostreatus IBL-02 MnP, both the free and immobilized MnP were used for the decolorization of four different textile industrial effluents. A maximum of 100% decolorization was achieved for the different textile effluents within the shortest time period. A lower KM , higher Vmax , hyper-activation, and enhanced acidic and thermal resistance up to 70 ℃ were the novel catalytic features of the sol-gel immobilized MnP, suggesting that it may be a potential candidate for biotechnological applications particularly for textile bioremediation purposes.

Validation of a serum microRNA panel as biomarkers for early diagnosis of hepatocellular carcinoma post-hepatitis C infection in Egyptian patients

AIM To investigate the prospective importance of serum micro(mi)RNAs(mi R-125 b, mi R-138 b, mi R-1269, mi R-214-5p, mi R-494, mi R375 and mi R-145) as early biomarkers for the diagnosis of hepatitis C virus(HCV)-related hepatocellular carcinoma(HCC).METHODS Two-hundred and fifty HCV4 a patients, 224 HCV4 aHCC patients, and 84 healthy controls were enrolled in the study. Expression levels of mi R214-5p, mi R-125 b, mi R-1269 and mi R-375 were quantified using quantitative real-time PCR.RESULTS Expression of the selected mi RNAs in serum wassignificantly lower in HCC patients than in the healthy controls, except for mi R-1269 and mi R-494. There was a significant difference between HCC and HCV patients, in particular for HCC and late stage fibrosis, rather than HCV patients and early fibrosis. It is obvious that mi R-1269 was significantly upregulated in HCC cases compared to hepatic fibrosis cases. Each mi RNA can show HCC progression. Multivariate logistic regression analysis indicated that the tested panel of mi RNAs(mi R214-5p, mi R-125 b, mi R-1269 and mi R-375) represent accurate and specific indictors of HCC development.CONCLUSION This study presents a panel of mi RNAs with strong power as putative diagnostic and prognostic biomarkers for HCV-induced HCC. Moreover, mi R-214-5p and mi R-1269 could be considered as early biomarkers for tracking the progress of liver fibrosis to HCC.

Tests to measure the quality of spermatozoa at spermiation

This commentary is to critique the revised World Health Organization （WHO） semen analysis manual as it pertains to characteristics of a spermatozoon at spermiation. The aims of the revised WHO manual include improving the ＇quality of semen analysis＇ without any restriction to clinical use. Furthermore, the manual states that semen analysis may be useful for （a） ＇investigating male fertility status＇ and （b） ＇monitoring spermatogenesis during and following male fertility regula- tion.＇ However, if the analysis of ejaculated spermatozoa is intended for the purposes described in （b）, then cells that are abnormal at spermiation must be identified. This paper takes the position that the manual does not identify methods to estimate the quality of spermatozoa at spermiation. Instead, it uses a ＇gold standard＇ of sperm passing through the cervical mucus or arriving near the site of fertilization. Although this standard is appropriate for drawing conclusions regarding the probability that an individual could impregnate his partner, it is not appropriate for studying illness of the testes per se. Herein, the measures of sperm quality presented in the WHO manual are critiqued with respect to the detection of spermatozoa that were abnormal at spermiation vs. those that became abnormal subsequently. Quality assessments based on the percentage of motile or ＇viable＇ spermatozoa are meaningless. Alternative quality attributes defining spermatozoa at spermiation are presented in this paper. In conclusion, assessment of spermatozoal quality at spermiation, on the basis of quality attributes of individual ejaculated spermatozoa, is best achieved through application of （a） a new paradigm for the morphological evaluation of sperm quality and （b） modern analytical techniques to evaluate, in an adequate sample, several appropriate independent attributes in each spermatozoon in order to more accurately identify the proportion of abnormal spermatozoa.