Twenty-three crossbred ewes were utilized during 1995, to investigate the possibility of xenogenous capacitation, using stallion spermatozoa. Ewes were grouped according to endocrine status as anestrus (n = 12) or estrus (n = 11) and were surgically inseminated with either fresh (FR), cooled/extended (FCE) or frozen/thawed (FZ) spermatozoal samples. The capacitation response of spermatozoa recovered from oviductal flushing 4 - 6 hours post-insemination, was assessed using a chlortetracycline (CTC) fluorescent probe. Data were recorded as percentages of spermatozoa exhibiting CTC staining patterns characteristic of capacitated (PCAP), unreacted (PUR) and acrosome reacted (PAR) sperm. Mean PCAP was not significantly different in estrous ewes despite an increasing trend. Mean PUR and PAR were also not different. Differences in capacitated, acrosome reacted and unreacted spermatozoa in inseminated and recovered samples (CAPDIF, ARDIF and URDIF, respectively) were analyzed. CAPDIF was significantly different between treatment groups (p < 0.01). CAPDIF was also significantly greater for the estrus versus anestrus group (p < 0.05). Total number of spermatozoa recovered (RTOTAL) was recorded. More spermatozoa were recovered from estrus ewes, however significance was not established. Mean number of spermatozoa recovered was 37.9 ± 35.9 per ewe. Treatment significantly affected RTOTAL in estrus animals (p = 0.01). FR samples had the highest recovery. Results suggest that xenogenous capacitation of stallion semen may occur in the reproductive tract of the ewe. Implications of these results are discussed in regards to their application of xenogenous gamete intrafallopian transfer (X-GIFT) as a treatment option for certain infertility problems in the mare. / Master of Science

Further in vivo functional studies were also performed. The SLC26A3 antibody was injected into the BALB/C mice seminiferous tubules using micropipette. The animals were sacrificed after three days, and CASA, daily sperm production (DSP) were used to evaluate sperm motility and spermatogenesis. The results showed that sperm motility was increased while there was no significant difference between DSP. Our results indicate that SLC26A3 on sperm does not play a dominant role in spermatogenesis, epididymal maturation and sperm motility. / In the first part of study, guinea pig sperm which were incubated in medium with various concentrations of Cl- resulted in varied percentages of capacitated sperm, in a concentration dependent manner. Depleting Cl-, even in the presence of HCO3 -, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO 3- dependent events, including cAMP production, protein tyrosine phosphorylation and pHi increase also depend on Cl - concentrations. Similar Cl- dependence was observed for sperm hyperactivated motility and sperm-egg fusion. The capacitation-associated events could also be significantly reduced by inhibitors or antibodies of CFTR and SLC26A3, with a more potent effect observed for niflumate, an inhibitor more selective for SLC26A3, over that of DIDS, an inhibitor more selective for SLC4 exchangers. The expression and localization of CFTR and SLC26A3 in guinea pig sperm were also demonstrated using immunostaining and Western blot analysis. Our results indicate that Cl- is required for the entry of HCO3- necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3 - with CFTR providing the recycling pathway for Cl- . / In the second part of study, GC-1 spg cell line that expresses SLC26A6 but not SLC26A3 was used as a negative control. The cells and sperm were pretreated with anion exchanger inhibitors and SLC26A3 antibody, and then membrane potential and intracellular calcium were measured. Our results showed that DIDS could inhibit the HCO3- deficiency induced depolarization of GC-1 spg cells as well as the depolarization induced by Cl- or HCO3- deficiency in sperm. Niflumate could inhibit the HCO3- induced [Ca 2+] i increase of the sperm but not GC-1 spg cells. SLC26A3 antibody had no effect on the GC-1 spg cells but it could block the depolarization caused by C--deficiency in sperm. / Our previous study has demonstrated the involvement of Cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate necessary for sperm capacitation. However, whether its involvement is direct or indirect remains unclear. The present study is design to investigate: (1) the possibility of a Cl-/HCO3- exchanger, solute carrier family 26, number 3 (SLC26A3), operating with CFTR during sperm capacitation, (2) the role and the underlying mechanisms of SLC26A3 in other sperm post-testicular processes and spermatogenesis. / Taken together, our results demonstrate the involvement of SLC26A3 in sperm function, particularly in transporting HCO3- necessary for sperm capacitation, which appears to be working with CFTR providing the recycling pathway for Cl- in parallel. The present results also provide an explanation to the observed subfertility in patients with SLC26A3 mutations. Further in vitro and in vivo studies also have shown that SLC26A3 does not play a predominant role in spermatogenesis but may affect other post-testicular maturation processes. / Chen, Wenying. / "November 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Includes bibliographical references (leaves 232-257). / xv, 257 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to determine if the compatible solutes (proline, glycine betaine, and trehalose), the epididymal compounds (taurine, hypotaurine and inositol) or the antioxidants (carnosine and ascorbic acid) in tris-citrate based diluents could improve the post-thaw survival and/or fertility of ram spermatazoa. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1996?

Thesis (MScMedSc (Obstetrics and Gynaecology))--University of Stellenbosch, 2005. / Chapter 1 contains a review dealing with nuclear and morphological changes during spermatogenesis and spermatozoa transport with emphasis on the maturation of spermatozoa, capacitation, acrosome reaction and the interaction with the cumulus oophorus complex (COC). The oocyte and cumulus oophorus complex is also discussed particularly on the topic of maturity (oocyte and cumulus maturity). Also presented is a review of the fluorescent binding agents, namely Fluorescein Isothiocyanate labeled with Pisum sativum (FITC-PSA), Chlorotetracycline test (CTC) and Chromomycin A3 (CMA3). Chapter II describes all the materials and methods used during this study. Routine semen analysis is described with emphasis on normal spermatozoon morphology according to strict criteria. The evaluation of capacitation and acrosome reaction (AR) using the CTC and PSA-FITC staining methods as well as the evaluation of spermatozoon nuclear chromatin packaging using the CMA3 staining method is described. Chapter III represents the results recorded in this study. Compared with those spermatozoa cultured in medium alone, spermatozoa exposed to the cumulus mass were more likely to be capacitated and acrosome reacted, with a distinct increase in chromatin packaging quality. A general discussion of the results and future applications are discussed in Chapter IV. In short An in vitro model for spermatozoa penetration through the cumulus oophorus was established. The model can be applied to investigate the effect of the cumulus oophorus on sperm functions and to assist in the selection of functional sperm for intracytoplasmic sperm injection therapy. All relevant references are presented in Chapter V .

Thesis (MSCMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / In this study, we presented data on the role of free radicals in human spermatozoa, particularly in the context of centrifugation and the potential development of defective sperm function. In order to achieve this, methods were developed to directly measure intracellular free radicals in human sperm and the effects of exogenously applied free radicals on sperm function were established. The role of brief and prolonged centrifugation and the associated generation of free radicals was also investigated.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The application of assisted reproductive biotechnologies in sheep flocks is hampered by the susceptability of ovine sperm to cryodamage. There is still considerable scope in the improvement of cryopreservation protocols for ovine sperm to minimize the degree of damage to sperm during the cryopreservation process. Pre-cryopreservation processing has a definite effect on the survivability, motility, and fertilizing ability of sperm. Little information is however available on the potential contribution of the genetic make-up of rams, divergently selected for fecundity, on the ability of sperm to offer resistance to the damage caused by cooling, cryopreservation and thawing. The study aimed to investigate the influence of genetic selection for prolificacy (i.e. High Merino Line and Low Merino Line in terms of fecundity) on the ability of ovine sperm to offer resistance to cryodamage. The study investigated the effect of pre-cryopreservation processing by comparing motility and morphometry traits recorded for fresh- and post-thaw Merino ejaculated and epididymal sperm samples obtained form the High and Low lines, respectively. The effect of different sperm concentrations, equilibration periods and the addition or omission of seminal plasma from cryopreserved samples on the viability and morphometrical traits were also investigated. Ejaculate samples were collected by means of the artificial vagina (AV) method from 8 High Line rams and 7 Low Line rams. Epididymal samples were collected from 6 rams of each of the High and Low lines respectively, by recovering the epididymal sperm via aspiration from the cauda epididymides post mortem. Ejaculate samples were subjected to macroscopic and microscopic evaluation, and epididymal samples only to microscopic evaluation, for which the Sperm Class Analyzer® program was used for the evaluation of motility and morphometric measurements. Sperm motility recordings were captured at 100 frames per second. From findings of the study, it was concluded that genotype had no positive influence on the conception rate of the ewes mated to the High or Low Line rams, even though the rams from the two lines differed significantly in terms of their serving capacity. When sperm morphometry was evaluated for fresh ejaculate samples, the two lines differed significantly in terms of the morphometric traits elongation and ellipticity. Epididymal and ejaculated sperm obtained from Low Line rams had broader and rounder heads, compared to sperm obtained from High Line rams. When morphometry was assessed for sperm samples between the two methods of sperm recovery (collected with an AV or recovery via aspiration from the cauda epididymides of sacrificed rams), no morphometrical differences were observed. Significant differences were reported for the majority of the sperm motility traits (i.e. percentage motile, rapid-, medium-, slow swimming, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of the lateral head displacement (ALH)) recorded for ejaculated and epididymal sperm. The motility traits ALH and beat-cross frequency (BCF) analysed for epididymal sperm differed significantly between the two lines. When epididymal sperm were evaluated post-thaw, it became evident that the sperm obtained from the High Line rams had a larger acrosome surface cover when compared to that of the Low Line ram sperm. The addition of seminal plasma to epididymal samples did not result in an improvement of the preservation of sperm motility. It is known from the literature that cryopreservation causes a decrease in sperm head size. Head width was unaffected by cryopreservation with the addition of seminal plasma in this study, indicating a potential benefit with the use of seminal plasma in the cryopreservation protocol of epididymal ram sperm. The study compared two pre-processing techniques, i.e. the more time consuming swim-up technique (SUT) with a more time-efficient ‘flush technique’ (FT) to optimize the pre-processing protocol for motility assessment of sperm samples before cryopreservation of ram sperm. Comparison of the SUT and FT indicated that almost all of the motility parameters measured using the FT compared favourably with those obtained using the SUT. The results indicated that the FT can be used a more time-efficient technique to use for determining the motility of a sperm sample prior to cryopreservation. In conclusion, line differences associated with reproduction were observed in terms of the serving capacity of the rams, with selection for fecundity influencing the morphometric traits elongation and ellipticity for sperm obtained from the two lines. Future studies should be aimed at investigating morphometric traits of ovine sperm, to correlate it with fertilizing ability of sperm post-thaw and ensure optimal cryopreservation processing. / AFRIKAANSE OPSOMMING: Die toepassing van ondersteunende reproduksie tegnieke in skaaptroppe word bemoeilik deur die onvermoë van ram sperme om weerstand teen bevriesingskade te bied. Daar is nog baie ruimte vir die verbetering van die bevriesingsprotokolle vir skaap sperm om die omvang van bevriesingskade te verminder. Voor-bevriesing verwerking het dan 'n besliste uitwerking op die oorlewing, beweeglikheid en bevrugtingsvermoë, van skaap sperme. Min inligting is beskikbaar oor die potensiële bydrae van die genetiese samestelling van ramme wat uiteenlopend op grond van vrugbaarheid geselekteer is, op die vermoë van skaap sperme om weerstand te bied teen die skade wat deur verkoeling, diepbevriesing en ontdooiing, veroorsaak word. Die doelwit van die studie was om die invloed van genetiese seleksie vir fekunditeit (d.i. Hoë Merino Lyn en Lae Merino Lyn in terme van fekunditeit) op die vermoë van skaap sperme om weerstand teen bevriesingskade te bied, te ondersoek. Die studie het getoets wat die bevriesing proses se effek op epididimale sperme is, deur sperm motiliteit en -morfometrie te vergelyk tussen vars gekollekteerde sperme en sperm monsters na ontdooiing. Die effek van verskillende sperm konsentrasies, ekwilibrasie tydperke en die byvoeging of uitsluiting van seminale plasma op die lewensvatbaarheid en morfometriese eienskappe van Merino ramsperme is ondersoek in die studie. Geëjakuleerde monsters is versamel met behulp van 'n kunsmatige vagina (AV) van 8 Hoë Lyn en 7 Lae Lyn ramme. Epididimale monsters is verkry van 6 ramme van elk van die Hoë en Lae Lyne, deur middel van aspirasie van die sperme uit die cauda epididimii nadoods. Geëjakuleerde sperm monsters is met behulp van makroskopiese en mikroskopiese metodes geëvalueer, en epididimale sperm monsters slegs mikroskopies geëvalueer, met behulp van die Sperm Class Analyzer® program wat vir die evaluasie van beweeglikheid en morfometriese afmetings gebruik is. Sperm beweeglikheids opnames is opgeneem teen 100 raampies per sekonde. Die resultate van die studie het aangedui dat genotipe geen effek het op besetting van die ooie gepaar met die Hoë of Lae Lyn ramme gehad het nie, terwyl die dekvermoë aansienlik tussen ramme van die twee lyne verskil het. Wanneer die morfometriese eienskappe van vars geëjakuleerde sperme vergelyk was, het die lyne beduidend in terme van die morfometriese eienskappe van verlenging (elongation) en elliptisiteit verskil het. Die epididimale en geëjakuleerde sperme verkry vanaf die Lae Lyn ramme het ʼn breër en ronder kopvorm getoon as sperme wat verkry is van die Hoë Lyn ramme. Wanneer die morfometriese eienskappe van sperme versamel met die twee verskillende metodes (d.i. kunsmatige vagina of aspirasie vanuit die cauda epididimides) vergelyk was, is geen morfometriese verskille waargeneem nie. Die meeste sperm beweeglikheidseienskappe (d.i. persentasie beweeglike, vinnig-, medium- en stadig-swemmende sperme, VCL, VSL, VAP en ALH) van geëjakuleerde en epididimale sperme het verskil. Die beweeglikheidseienskappe amplitude van die laterale verplasing van die spermkop (ALH) en frekwensie waarmee sperm sy eie pad kruis (BCF), soos bepaal vir epididimale sperme, het beduidend tussen die twee lyne verskil. Met die evaluering van epididimale sperme na ontdooiing was dit duidelik dat sperme verkry van die Hoë Lyn ramme 'n groter mate van akrosoom-oppervlak gehad het, in vergelyking met sperme van die Lae Lyn ramme. Die byvoeging van seminale plasma by epididimale monsters het nie bygedra tot 'n verbetering van spermbeweeglikheid nie. Bestaande literatuur dui aan dat diepbevriesing 'n afname in die kopgrootte van sperme veroorsaak. In hierdie studie het die byvoeging van seminale plasma ʼn verandering in kopgrootte voorkom, wat dui op ʼn potensiële voordeel om seminale plasma in die bevriesingsprotokol van epididimale ramsperme in te sluit. Die studie het twee beweeglikheid bepalingstegnieke vergelyk om te bepaal of die tydrowende “opswem” tegniek (SUT) vervang kan word met 'n meer tyd-doeltreffende "spoel tegniek” (FT) in die voorbevriesing verwerking protokolle van ram sperme. Vergelyking van die twee tegnieke het aangedui dat die meeste van die kinematiese eienskappe van die FT gunstig met die waardes soos verkry met die SUT, vergelyk het. Resultate het getoon dat die FT parameters goed vergelyk met die beweeglikheid parameters van die SUT, dus kan dit aangeneem word dat die FT ʼn meer tyd-doeltreffende tegniek is wat vergelykbare sperm beweeglikheidsinligting oor skaap sperm monsters voor bevriesing sal verskaf. In samevatting is verskille in terme van die dekvermoë en op morfometriese vlak, meer spesifiek die eienskappe van verlenging (elongation) en elliptisiteit, tussen die twee lyne waargeneem. In toekomstige studies moet die morfometriese eienskappe van skaapsperme verder bestudeer word, asook die korrelasie daarvan met die bevrugtingsvermoë na ontdooiing bepaal om sodoende die diepbevriesing protokolle van skaapsperme te optimaliseer.

Thesis (D.Phil.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme. / AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.

In externally fertilizing fishes, multiple factors of the spawning environment may affect the sperm viability, and thus the fertilization rate. In this thesis, the sperm activation effect of osmolality of non-electrolytes and electrolytes activation media, pH and ion channel inhibitors on Nile tilapia, Oreochromis niloticus, and the effect of environmentally relevant pollutants (cadmium, malathion and rotenone) on sperm fitness (motility and morphology) were investigated. Seminal fluid samples collected from male fishes (200-250g) were subjected to activation treatments, then analyzed for sperm motility using motility score, and motility variables using Hobson sperm tracker for straight line velocity (VSL), beat cross frequency (BCF) and percentage of motile cells (MOT). For the ion channel inhibitors and pollutants, the effect on sperm motility variables of VSL, VCL (curvilinear velocity) and LIN (linearity) were determined. Multivariate analysis was also carried out to determine the effects of ion channel inhibitors and pollutants on sperm subpopulations. The effects of pollutants on sperm morphology were observed using microscopy techniques, namely, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Sperm motility was initiated when the sperm were exposed to hypoosmotic electrolytes and non-electrolytes solution. We also found that sperm show optimal activity at pH range of 6-8 which depicts that the effect of pH on sperm motility is negligible. Lanthanum (calcium channel blocker) and flunarizine (sodium-calcium exchanger pump blocker) were found to inhibit sperm motility at 25 and 5 µM, respectively, suggesting that both ion channels play a significant role in sperm activation in O. niloticus. In contrast amiloride, ouabain and quinine showed no effects on activation, indicating that epithelial sodium channels, sodium-potassium ATPase and voltage gated potassium channels respectively are unlikely to have major roles in sperm activation or motility. The spermatozoa of Oreochromis niloticus were uniflagellate with clearly differentiated oval-shaped head, midpiece and flagellum. Sperm exposed to hypoosmotic shock showed swelling of the midpiece and sleeve structure. The pollutants showed dose- and time-dependent effect on sperm motility of the fast linear sperm subpopulation. Sperm morphology was not affected. Sperm motility was inhibited at 0.44, 0.03 and 0.063 µM, cadmium, malathion and rotenone respectively. Both cadmium and malathion exerted effects very quickly after exposure. The effect of cadmium, which can exert toxicity by calcium antagonism, is consistent with the effects of calcium channel blockes and further supports an important role for calcium in sperm activation and motility. Malathion had effects at relatively low, environmentally relevant concentrations, suggesting the presence of functionally important acetylcholinesterase activity in sperm, and also the presence of activation cytochrome P450 activity. Rotenone, a well known mitochondrial poison, affected motility only after 15 min of pretreatment. The alteration of sperm trajectories in fast linear spermatozoa subpopulation by pollutants at submicromolar concentrations as demonstrated in our study implies potentially serious consequences for fish populations in polluted environments. Furthermore the results indicate that fish sperm motility as assessed by CASA could be an ecologically relevant, sensitive, and ethically acceptable method for toxicity testing in environmental risk assessment.

A preservação do sêmen suíno refrigerado para fins de inseminação artificial é a forma mais utilizada no mundo. Para tal, os espermatozoides são diluídos em meios que forneçam substratos para nutrir e proteger estas células. Porém, a faixa de temperatura de armazenamento do sêmen suíno refrigerado não é capaz de cessar completamente os mecanismos metabólicos dos espermatozoides, que em ambiente aeróbico da dose inseminante, produzem e liberam continuamente produtos do metabolismo do oxigênio. A ação das espécies reativas de oxigênio sobre o espermatozoide é uma das razões do declínio na população de células espermaticas estrutural e funcionalmente aptas à fertilizarem os gametas femininos. Portanto, a adição de um composto antioxidante ao meio diluidor poderia melhorar ou manter constante o número de espermatozoides viáveis ao longo do período de conservação da dose inseminante. Neste contexto, este trabalho objetivou verificar se a adição do antioxidante resveratrol geraria modificações positivas em parâmetros celulares relacionados à qualidade do sêmen suíno refrigerado à 15-17°C avaliados por sistema computadorizado da motilidade espermática e citometria de fluxo (experimento in vitro), e se este antioxidante melhoraria os índices de fertilidade na inseminação artificial intrauterina (IAIU) através da recuperação, contagem e identificação dos embriões (experimento in vivo). As concentrações testadas no experimento in vitro não superaram os resultados do tratamento controle (p&lt;0,05), sendo a concentração de 1,0 mM prejudicial ao espermatozoide suíno (p&lt;0,05). A utilização da concentração de 0,01 mM de resveratrol para inseminação das fêmeas suínas no experimento in vivo não apresentou efeito positivo nos índices de taxa de prenhez e fertilidade ajustada total (p&gt;0,05), além de ter resultado em uma baixa taxa de embriões viáveis (p&lt;0,05), quando comparados ao tratamento controle. Deste modo, pode concluir que não é indicado a adição do antioxidante resveratrol ao meio diluidor para inseminação artificial, uma vez que compromete a qualidade das células espermáticas do reprodutor suíno e impacta negativamente na fertilidade das fêmeas. / The preservation of chilled boar semen for artificial insemination is the most performed worldwide. For this, the sperm are extended in medium that provides substrates to nourish and protect these cells. However, the storage temperature range of chilled semen is not able to completely stop the metabolic mechanisms of sperm, which keeps producting and releasing oxygen reactive products in aerobic environment of insemination doses. The action of reactive oxygen species on the sperm is one of the reasons for decreasing sperm population with structural and functional capacity to fertilize the female gamete. Therefore, the addition of antioxidant compound to the extender medium could improve or maintain the number of viable spermatozoa throughout the conservation time of insemination doses. In this context, this work aimed to determine whether addition of antioxidant resveratrol would generate positive changes in cell parameters related to the quality of boar semen cooled to 15-17 °C assessed by computered analysis of sperm motility and flow citometry (in vitro experiment) and whether this antioxidant would improve fertility rates using intrauterine insemination (IUI) by the recovery, counting and embryos identification (in vivo experiment). The concentrations tested in vitro experiment have not exceed control treatment results (p &lt;0.05), with the concentration of 1.0 mM being detrimental to the boar spermatozoa (p &lt;0.05). The concentration of 0.01 mM of resveratrol used for gilts insemination in experiment in vivo has not shown positive effect on pregnancy rate and the total adjusted fertility (p&gt; 0.05) and have resulted in a low rate viable embryos (p &lt;0.05) compared to control. Thus, we conclued that the addition of resveratrol antioxidant in boar extender medium is not suitable for artificial insemination, once it compromises the quality of boar sperm cells and impairs on female fertility.

O presente trabalho foi composto de dois estudos distintos. O Estudo I, com um foco conservacionista, teve como objetivo a avaliação e comparação de diferentes métodos de criopreservação de sêmen de bugio-preto (Alouatta caraya). Para tanto, o estudo foi dividido em dois experimentos: Experimento I composto de dois ensaios no primeiro ensaio foram comparados dois diluidores comerciais BotuBOV e Test-yolk buffer (TYB) e no segundo ensaio foram comparados dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação dos efeitos da adição de DHA e de Trolox (análogo da vitamina E) ao diluidor para criopreservação de sêmen. O diluidor TYB apresentou melhores resultados quando comparados ao BotuBOV. A congelação lenta apresentou melhores resultados quando comparada à vitrificação. Não houve diferença entre o diluidor controle e os diluidores com Trolox, DHA ou combinação dos dois (DHAT), com exceção da integridade de acrossoma, que foi significativamente menor para o diluidor DHAT. Conclui-se que são necessários mais estudos, com utilização de outras doses de DHA e Trolox, além de outros antioxidantes. O Estudo II, com foco em pesquisa biomédica, teve como objetivo a criopreservação de sêmen de macaco-rhesus (Macaca mulata) e avaliação da qualidade pós-descongelação por meio de fecundação in vitro (FIV). Para tanto, o estudo foi dividido em três experimentos: Experimento I avaliação e comparação de dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação e comparação de quatro métodos de preparação do sêmen pós-descongelação lavagem simples (LS), swim-up (SU), separação por gradiente de densidade (SGD) e filtragem em lã de vidro (FLV); Experimento III avaliação da qualidade seminal pós-descongelação por meio de FIV. A congelação lenta apresentou melhores resultados que a vitrificação (p<0,05). LS apresentou os melhores resultados, seguido por SGD e SU, enquanto FLV apresentou os piores resultados. LS e SGD foram utilizados para avaliação da qualidade seminal por meio de FIV, utilizando sêmen fresco como controle. As taxas de fecundação (média±EPM%) para oócitos MI inseminados com sêmen fresco (43.5±16.4) foram significativamente maiores (p<0,05) que LS (2.0±2.0), mas não diferiram de SGD (25.1±14.2). Não houve diferença na taxa de blastocistos (média±EPM%) entre os tratamentos (variação de 0 a 11.9±7.9). As taxas de fecundação para oócitos MII inseminados com sêmen fresco (41.4±3.6) também foram significativamente maiores que SGD e LS (18.4±6.9 e 12.7±7.7, respectivamente), assim como a taxa de blastocistos (64.7±13.6; 4.7±4.7; 30.9±13.8, respectivamente). Conclui-se que, espermatozoides criopreservados foram capazes de fertilizar oócitos e os embriões atingiram o estágio de blastocisto. A SGD selecionou espermatozoides pós-descongelação de melhor qualidade para FIV em macacos-rhesus, quando comparada à LS / This work was divided in two studies. The objective of Study I was to test and compare different cryopreservation methods for sperm from black-and-gold howler monkeys (Alouatta caraya), with a focus on species conservation. The study was divided in two experiments. Experiment I composed of two trials the first trial compared two commercial extenders BotuBOV and Test-yolk buffer (TYB), and the second trial compared two cryopreservation methods slow freezing and vitrification; Experiment II evaluation of the effects of DHA and Trolox (vitamin E analog) as additives to the freezing extender. TYB had better results when compared to BotuBOV. Slow freezing had better results when compared to vitrification. There was no difference between control extender (TYB) and extender containing Trolox, DHA or a combination of both (DHAT), except for acrosome integrity, which was significantly lower for DHAT. In conclusion, more studies are necessary, using other doses of DHA and Trolox, as well as other antioxidants. The objective of Study II was to assess the quality of frozen-thawed sperm from rhesus macaques (Macaca mulatta) by in vitro fertilization (IVF). The study was divided in three experiments. Experiment I evaluation and comparison of two cryopreservation methods slow freezing and vitrification; Experiment II evaluation and comparison of four preparation methods for frozen-thawed sperm simple wash (SW), swim-up (SU), density gradient centrifugation (DGC), and glass wool filtration (GWF); and Experiment III evaluation of frozen-thawed sperm quality by IVF. Slow freezing had better results when compared to vitrification (p<0,05). SW had better results, followed by DGC and SU, while GWF had the worse results. SW and DGC were further evaluated by IVF. Fertilization rates (mean±SEM%) with MI oocytes using fresh sperm were significantly higher (43.5±16.4) than with SW (2.0±2.0) and did not differ from DGC (25.1±14.2). There was no difference in blastocyst rates between treatments (range 0 to 11.9±7.9). Fertilization rates with MII ova were also significantly higher with fresh sperm (41.4±3.6) than DGC and SW (18.4±6.9 and 12.7±7.7, respectively), and more blastocysts developed from MIIs fertilized with fresh sperm (64.7±13.6) than SW and DGC (4.7±4.7 and 30.9±13.8, respectively). In conclusion, frozen-thawed sperm were able to fertilize oocytes and embryos reached the blastocyst stage. DGC yielded better frozen-thawed sperm for IVF in rhesus macaques, when compared with SW