Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

This thesis explores the far-reaching impacts of sexual conflict over care on a suite of traits in the penduline tit family (Remizidae), further confirming the intricate relationships between parental care, mating systems and sexual selection. The results reveal the first genetic phylogeny of this family and suggest that uniparental care evolved once in this group. The transition to uniparental care is associated with rapid evolution of male plumage ornaments most likely driven by increased sexual selection. The results also suggest a relationship between male care and the likelihood of paternity on an evolutionary time-scale, as the biparental species exhibit much lower levels of promiscuity than the uniparental European penduline tit. Increased promiscuity was also found to impact sperm morphology in the penduline tits and allies with greater sperm length uniformity in more promiscuous species. This sperm trait was also discovered to co-vary with a sexually selected plumage trait in the European penduline tit suggesting potential interactions between female mate choice and male fertility. An investigation into genetic diversity within the Sylvioidea super-family finds large variation but does not suggest any link between promiscuity and genetic diversity as predicted if promiscuity maintains a higher effective population size in these passerines. The work highlights the interlinked relationships between parental care, mating systems and sexually selected traits, which are increasingly studied in concert. The consequences of sexual conflict over care appear to be far-reaching in the penduline tits, however the degree to which they feedback upon each other and the effect that it has on speciation remains to be seen. The penduline tits further prove their ability to provide valuable insight into the evolution of sexual conflict.

Thesis (DSc)--Stellenbosch University, 2012. / Please refer to full text for abstract.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Tankwa goats have been free-ranging in the Tankwa Karoo National Park in the Northern Cape for more than 80 years. A genetic study concluded that these feral goats are a unique genetic resource compared to other goat breeds in South Africa and should be conserved as a distinctive population. A decision taken by the South African National Parks who is the managing authority in the park, was to remove all alien species, which included the Tankwa goats. Several animals were translocated to the Carnarvon Research Station by the Northern Cape Department of Agriculture, Land Reform & Rural Development, where the Tankwa goat population has grown to a few hundred individuals. Currently, sound scientific decisions including the application of a wide range of technologies and approaches are applied to conserve the population, such as an informed understanding of the reproductive biology of these goats. The aim of this study was to define sperm quality in Tankwa goats using various macroscopic and microscopic evaluation techniques.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-07T13:07:20Z No. of bitstreams: 1 Filipe Queiros Gondim Bezerra.pdf: 347211 bytes, checksum: 57418693331f5977fdbc1b87348c5c9e (MD5) / Made available in DSpace on 2016-10-07T13:07:20Z (GMT). No. of bitstreams: 1 Filipe Queiros Gondim Bezerra.pdf: 347211 bytes, checksum: 57418693331f5977fdbc1b87348c5c9e (MD5) Previous issue date: 2007-02-26 / This work had the objective to determine the puberty and the spermatic representation to the males puberty (n = 22) of the Boer breeding, being determined the concentration of testosterone in relation with body weight and scrotal perimeter from Day 30 to 300 of life between spermatic motility verses spermatic mass motility, spermatic vigor, defects bigger and smaller defects of the spermatozoa in the dry and rainy seasons. In the dry season these parameters had varied from 3,7±1,1 to 34,0±4,7 (body weight), from 7,9±0,8 to 25,7±2 (scrotal perimeter) and from 259,41±172,35 to 4613,41±2892 pc/mL (testosterone). The spermatic concentration varied from 0,96±0,60 x 109/mL to 2,15±0,73 x 109/mL, the forward individual motility from 35,4%±31,2% to 64.1%±29.1%, the mass motility from 1,7±1,6 to 3,1±1,1, vigor from 2,0±1,6 to 3,0±1,5, bigger defects from 16,9%±8,9% to 4.2%±2.7% and smaller defects from 17,5%±5,7% to 3,2%±1,6%. In the rainy season ranged from 9,7±2,3 to 28,1±6,9 (body weight) from 9,5±1,5 to 22,0±3,0 (scrotal perimeter) and from 521,9±311,27 to 3417,9±2021,77 pc/mL (testosterone). The spermatic concentration diverse from 0,90±1,46 x 109/mL 2,7±1,65 x 109/mL, the forward individual motility from 16,0%±21% to 62.0%±34%, the mass motility from 0,3±0,9 to 2,6±1,7, vigor from 1,1±0,9 to 2,8±1,8, bigger defects from 18,3%±3,6 % to 3%±1.2% and smaller defects from 16,1%±6,2% to 3,4%±1,4%. It was also registered positive correlation between the concentration , testosterone and body weight (r = 0,30; r = 0,43), testosterone and scrotal perimeter (r = 0,42; r = 0,52) and between body weight and scrotal perimeter (r = 0,93; r =0,88) in the dry and rainy seasons, respectively. Also was registered positive correlation between spermatic motility versus spermatic mass motility (= 0,93; r = 0,93), spermatic motility versus spermatic vigor (r = 0,94; r = 0,92), as well as negative response between individual motility gradual versus bigger defects of the spermatozoa (r = -0,28; r = -0,25) and between forward individual motility and smaller defects of the spermatozoa (r = -0,21; r = -0,30), respectively, in the rainy and dries seasons. At the third month of age, 70.0% of the animals born in the rainy season had presented penis detachment from the prepuce , fact that only occurred in the fifth month of life in 67,6% of the animals been born in the dry season. At the four and seven months, respectively, the penis detachment from the prepuce occurred in all the born animals. The results allow to conclude that the dry season delays the unleash of the puberty, that the plasmatic concentration of testosterone is directly related with the puberty and body weight, scrotal perimeter and penis detachment from the prepuce, can be an important signify of the beginning of puberty, certified with base in a set of reproductive parameters in the young male Boer goat. / Este trabalho teve o objetivo de determinar a puberdade e o quadro espermático à puberdade de machos (n = 22) da raça Boer, determinando as relações da concentração de testosterona com peso corporal, perímetro escrotal dos 30 aos 300 dias de vida e as relações entre motilidade espermática versos turbilhonamento espermático, vigor espermático, defeitos maiores e defeitos menores dos espermatozóides nas estações seca e chuvosa. Na estação seca esses parâmetros variaram de 3,7±1,1 a 34,0±4,7 (peso corporal) de 7,9±0,8 a 25,7±2 (perímetro escrotal) e de 259,41±172,35 a 4613,41±2892 pc/mL (testosterona). A concentração espermática variou de 0,96±0,60 x 109/mL a 2,15±0,73 x 109/mL, a motilidade individual progressiva de 35,4%±31,2% a 64,1%±29,1%, o turbilhonamento de 1,7±1,6 a 3,1±1,1, vigor de 2,0±1,6 a 3,0±1,5, defeitos maiores de 16,9%±8,9% a 4,2%±2,7% e defeitos menores de 17,5%±5,7% a 3,2%±1,6%. Na estação chuvosa oscilou de 9,7±2,3 a 28,1±6,9 (peso corporal) de 9,5±1,5 a 22,0±3,0 (perímetro escrotal) e de 521,9±311,27 a 3417,9±2021,77 pc/mL (testosterona). A concentração espermática variou de 0,90±1,46 x 109/mL a 2,7±1,65 x 109/mL, a motilidade individual progressiva de 16,0%±21% a 62,0%±34%, o turbilhonamento de 0,3±0,9 a 2,6±1,7, vigor de 1,1±0,9 a 2,8±1,8, defeitos maiores de 18,3%±3,6 % a 3%±1,2% e defeitos menores de 16,1%±6,2% a 3,4%±1,4%. Foi registrada correlação positiva entre a concentração de testosterona e peso corporal (r =0,30; r =0,43), testosterona e perímetro escrotal (r =0,42; r =0,52) e entre peso corporal e perímetro escrotal (r =0,93; r =0,88) nas estações seca e chuvosa, respectivamente. Também foi registrada correlação positiva entre motilidade espermática vs. turbilhonamento espermático (r =0,93; r =0,93), motilidade espermática vs. vigor espermático (r =0,94; r =0,92), bem como correlação negativa entre motilidade individual progressiva vs. defeitos maiores dos espermatozóides (r = - 0,28; r = -0,25) e entre motilidade individual progressiva e defeitos menores dos espermatozóides (r = -0,21; r = -0,30), respectivamente, nas estações chuvosa e seca. Aos três meses de idade, 70,0% dos animais nascidos na estação chuvosa apresentaram desbridamento do pênis, fato que somente ocorreu no quinto mês de vida em 67,6% dos animais nascidos na estação seca. Aos quatro e sete meses, respectivamente, o desbridamento ocorreu em todos os animais nascidos nas duas estações. Os resultados permitem concluir que a estação seca retarda o desencadeamento da puberdade, que a concentração plasmática de testosterona está diretamente relacionada com a puberdade e que o peso corporal, perímetro escrotal e desbridamento do pênis, podem ser indicadores importantes do início da puberdade, certificada com base num conjunto de parâmetros andrológicos obtidos no macho caprino jovem da raça Boer.

No description available.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.

Sperm phenotype is an essential indicator of the male ejaculate quality and may have a significant impact on male reproductive success. Sperm phenotypes are considerably variable across species but variation is also found among males within species. This thesis examines (1) variation in sperm phenotypes among males in barn swallows (Hirundo rustica), (2) changes in male ejaculate quality with age, (3) relationships between sperm morphology and motility, (4) effects of sperm phenotypes (morphology and motility) on male fertilization success, using a large dataset of 174 observation for 130 males, and (5) the influence of female reproductive environment on sperm motility. From the tested variables, only midpiece length correlated with male age. Older males had shorter midpiece but no relationship between male age and reproductive success was found. Sperm length negatively affected sperm motility and, simultaneosly, relative midpiece length posively correlated with sperm motility. No correlation was found between the male reproductive success and sperm motility, presence of abnormalities, length of outermost tail feathers or age. Males with shorter relative midpiece were more successful in within-pair paternity, and males with shorter sperms but longer relative midpiece were more successful in...