Introduction. Sperm parameter analysis is the standard method of male fecundity testing; however, minimal evidence supports associations between individual sperm parameters and reproductive outcomes. Our previous work shows strong associations between sperm mitochondrial DNA copy number (mtDNAcn) and time-to-pregnancy (TTP) in general populations, and between mtDNAcn and fertilization outcomes in clinical populations. Thus it is possible for sperm mtDNA biomarkers to act as summary measures of semen quality. In this study, we developed a sperm quality index (SQI) from semen parameters and compared its ability to measure fecundity to sperm mtDNAcn. Methods. We received 384 semen samples from the Longitudinal Investigation of Fertility in the Environment Study. Sperm mtDNAcn and mtDNA deletions (mtDNAdel) were quantified using a triplex probe-based qPCR method. The SQI was developed by ranking and summing select sperm parameters within the study population, including sperm concentration, sperm count, normal morphology, high DNA stainability, and DNA fragmentation to create a cumulative index. Discrete-time proportional hazards models were used to determine fecundability odds ratios (FOR), indicating associations between mtDNAcn, SQI, and TTP. Receiver operating characteristic (ROC) analyses determined the validity of the SQI and mtDNAcn as predictors of pregnancy within 12 months. Results. The SQI was highly associated with mtDNAcn, both continuously (Spearman Rho: -0.487; p-value: <0.001) and in deciles (ANOVA p-value: <0.001). The SQI (FOR: 1.25; 95% confidence interval (CI): 1.09, 1.43) and mtDNAcn (FOR: 0.754; 95% CI: 0.657, 0.866) performed similarly in discrete-time survival models and indicated a significant decrease and increase in TTP, respectively. MtDNAcn more effectively predicted pregnancy within 12 months (AUC: 0.703; 95% CI: 0.617, 0.789) than the SQI (AUC: 0.642; 95% CI: 0.531, 0.753). With multiple predictors, mtDNAcn outperformed summary models, with addition of the SQI and percent normal morphology minimally increasing model efficacy (AUC: 0.718, 95% CI: 0.617, 0.819). Conclusion. The association between the SQI and mtDNAcn suggest that mtDNAcn may serve as a summary biomarker for overall sperm quality. Neither individual nor summed sperm parameters are useful indicators of couple fecundity and reproductive outcomes compared to mtDNAcn. These results suggest that mtDNAcn has potential for use as a biomarker of fecundity.

Orientador: Frederico Ozanam Papa / Resumo: Durante o processo de criopreservação os espermatozoides de garanhões sofrem danos osmóticos que são irreversíveis. Mesmo utilizando-se as melhores técnicas de criopreservação, este processo ainda é prejudicial às células espermáticas sendo em média a taxa de sobrevivência espermática em torno de 50%. Além disso, ainda há uma porcentagem de espermatozoides sobreviventes portadores de danos subletais, fazendo com que seja limitada a capacidade de sobrevivência das células espermáticas após a descongelação e como consequência existe uma queda na fertilidade. Está bem estabelecido que os espermatozoides de diferentes garanhões domésticos variam significativamente em criosensibilidade, este fato pode estar relacionado com o efeito do crioprotetor, assim como pode ter relação com o tamanho molecular do crioprotetor penetrante e curva de refrigeração utilizada. Enquanto o glicerol continua a ser o crioprotetor mais comum para espermatozoides, existem fatores limitantes pelo seu uso, incluindo danos na membrana através de um efeito osmótico, sendo assim, as amidas têm sido estudas com o objetivo de minimizar os efeitos causados pelo glicerol durante o processo de criopreservação, por apresentarem melhor crioproteção da membrana das células espermáticas. Sendo assim, a proposta desse trabalho foi avaliar diferentes crioprotetores em diferentes curvas de refrigeração sobre a qualidade do sêmen equino. Para tanto, o estudo foi realizado para avaliar o efeito dos crioprotetores glicerol... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: During the cryopreservation process, sperm from stallions suffer osmotic damage that is irreversible. Even using the best cryopreservation techniques, this process is still harmful to sperm cells, with an average sperm survival rate of around 50%. In addition, there is still a percentage of surviving sperm with sublethal damage, causing the sperm cells' ability to survive after thawing to be limited and as a consequence there is a fall in fertility. It is well established that sperm from different domestic stallions vary significantly in cryosensitivity, this fact may be related to the effect of the cryoprotectant, as well as it may be related to the molecular size of the penetrating cryoprotectant and the cooling curve used. While glycerol remains the most common cryoprotectant for sperm, there are limiting factors for its use, including damage to the membrane through an osmotic effect, so amides have been studied with the aim of minimizing the effects caused by glycerol during cryopreservation process, as they present better cryoprotection of the sperm cell membrane. Thus, the purpose of this work was to evaluate different cryoprotectants in different refrigeration curves on the quality of equine semen. To this end, the study was carried out to evaluate the effect of cryoprotectants glycerol, methylformamide, dimethylformamide and dimethylacetamide in concentration at 5% in four refrigeration curves: 1°C/min-1, 0.25°C/min-1, 4°C/min-1 with stabilization at 5°C for 15min and... (Complete abstract click electronic access below) / Doutor

MSCAGR (Animal Science) / Department of Animal Science / The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat buck semen. Six ejaculates were collected from six indigenous bucks by means of electro-ejaculator method, and semen was pooled, and replicated 10 times. Raw semen were randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. All six groups were analysed for spermatozoa motility rates using computer-aided sperm analysis (CASA). The spermatozoa viability for all groups were assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Both the Triladyl® and Bioxcell® washed semen groups were diluted (1:4) with Phosphate Buffered Saline (PBS) then centrifuged at 1500 x g for ten min and seminal plasma was aspirated using 1 mL sterile plastic pipette. Semen samples were diluted (1:4) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5 ºC for 2 hours. Following equilibration, semen parameters were analysed. Thereafter, the semen samples were loaded into straws and placed 5 cm above a liquid nitrogen vapour for 10 min, and then stored at -196 ºC until use. Following one month of storage, frozen semen straws per treatment group were thawed at 37 ºC for 30 seconds, then semen parameters were analysed again. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. Total Spermatozoa motility rate of Bioxcell® (92.5±4.6), (68.2±13.5) and Triladyl® (94.9±5.5), (63.1±15.1) were significantly reduced (P < 0.05) following equilibration and freeze-thawing process, respectively on washed semen groups. Live and normal spermatozoa percentages were drastically reduced in Bioxcell® (5.2±4.9) and Triladyl® (6.9±8.6) washed semen groups, following freeze-thawing. There was a significantly lower number of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Triladyl® (68.7±26.8) compared to non-washed semen extended with Bioxcell® (49.8±20.1) following the freeze-thawing process. In conclusion, the freezing-thawing process did reduce the indigenous buck semen parameters irrespective of removal or non-removal of seminal plasma. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of buck semen.

MSCAGR (Animal Science) / Department of Animal Science / See the attached abstract below

<p>In recent years conservation of minor livestock breeds has been faced with numerous challenges attributed to decreasing national herd sizes, as well as differences in reproduction and growth. One such minor swine breed, the Large Black pig (LB), is increasingly attractive to small farmers due to their foraging abilities and carcass characteristics. Therefore, the LB pigs have been used in niche pork production systems which market pasture-raised pork products. The LB breed is critically endangered, maintaining a registered breeding population of less than 400 animals, with increasing prevalence of inbreeding and genetic drift. Therefore, the LB breed could benefit from a genetic importation to increase genetic diversity in a national herd with rapidly decreasing animal numbers. A genetic importation would require frozen semen to be brought in from another country for use in breeding U.S. pigs. Frozen-thawed semen (FTS) presents challenges for swine due to the reduced motile sperm cells which negatively impacts fertility. Therefore, the present study evaluated the utilization of FTS in a genetic importation for the LB pig. </p><p>A genetic importation occurred in 2016 where semen from the United Kingdom was used on various farms in the U.S. but resulted in zero piglets born. Therefore, 16 LB sows were donated to Purdue University for research into improving estrous and ovulation synchronization to facilitate FTS utilization. Four breeding replicates were performed where following 14 days of Matrix feeding, OvuGel® was administered at 144 h following last Matrix feeding (LMF) or 96 h in post-weaned sows and two FTS inseminations occurring at: 30 and 36 h, 17 and 23 h, 24 and 30 h, and 24 and 32 h after OvuGel® for replicates 1-4, respectively. Approximately 2.64±0.3 billion motile sperm cells per insemination were utilized in replicates 1-3 with American LB FTS, with replicate 4 utilizing 0.34±0.03 billion motile sperm cells of imported FTS. Follicle diameter (<i>P</i>=0.260), ovulation within 48 h of OvuGel® (<i>P</i>=0.411), and weight prior to breeding (<i>P</i>=0.681) did not influence conception rate, however expression of estrus was determined to significantly influence conception rate (<i>P</i>=0.043). Seventy-five LB piglets were weaned across the first three breeding replicates, with parity 2 sows observed to have larger litter sizes than parity 1 sows (<i>P</i>=0.066).</p> <p>Large Black and Duroc-sired (DS) crossbred pigs from replicates 1 and 2 farrowing were fed corn and soybean meal based finishing diets supplemented with (FIB) or without alfalfa and wheat middlings (CON). Following 6 dietary phases through finishing, 25 LB and 25 DS pigs were slaughtered at similar ages for digestive organ dissection and carcass measurements. Loin muscles were evaluated for fresh pork quality and instrumental color and tenderness. LB pigs had a reduced ADG (<i>P</i><0.0001) and G:F (<i>P</i><0.0001) compared to DS pigs. Pigs fed FIB resulted in reduced ADG (<i>P</i>=0.020) and reduced G:F (<i>P</i>=0.007). At slaughter LB pigs were 26.4 kg lighter than DS pigs (<i>P</i><0.0001), and pigs that were fed FIB had lighter live weights (<i>P</i>=0.002) than pigs fed CON. LB pigs had 28.5±1.3 cm² smaller longissimus muscle area (<i>P</i><0.0001), yielding 2.0 cm more 10^th rib back fat than DS pigs (<i>P</i><0.0001). CON pigs had heavier HCW (<i>P</i><0.0001) than FIB pigs, however FIB pigs had greater percent lean (<i>P</i>=0.015). LB pigs had significantly reduced percent lean than DS pigs (<i>P</i><0.0001). LB pigs had loins with reduced drip loss (<i>P</i>=0.009) and cooked shear force values (<i>P</i><0.0001). Overall, the growth and carcass composition of the pigs was most affected by genotype, and to a lesser extent than the type of diet fed. </p> <p>In conclusion, the genetic importation of LB semen was successful as ½ blood piglets were created for dispersal into the U.S. LB herd. Improvements in FTS utilization in this heritage breed contributed to the successful creation of live-born pigs. Additionally, growth and carcass information was obtained for LB breeders to use in understanding and marketing of this heritage breed of pigs.</p>

Tesis por compendio / [ES] Cualquier mejora en el conocimiento de la nutrición y el manejo de los futuros conejos reproductores debe considerarse como prioritario en la producción cunícola. Sin embargo, los principales esfuerzos en investigación se han centrado en estos animales durante la fase de reproducción propiamente dicha. La hipótesis inicial de esta tesis fue que alcanzar un desarrollo temprano adecuado durante la gestación, lactancia, engorde y recría, con la ayuda de programas ajustados de alimentación en algunos de estos períodos, mejoraría el rendimiento reproductivo y la vida útil de los futuros conejos reproductores. Para evaluar esta hipótesis, se llevaron a cabo dos tipos de ensayos, tanto en conejos hembra de líneas maternas como en machos de líneas seleccionadas por velocidad de crecimiento y destinadas a la inseminación artificial. El primero de estos ensayos se centró en los diferentes patrones de crecimiento, desde el nacimiento hasta el final de la fase de recría, y su influencia en la carrera reproductiva y la vida útil del conejo. En el segundo tipo de pruebas, se evaluó el efecto del uso de diferentes dietas o programas de alimentación durante el período de recría en la productividad a corto y medio plazo. Los resultados muestran dos fases importantes en el desarrollo temprano, de acuerdo con sus efectos sobre el rendimiento reproductivo y la vida útil. Mostrar un mayor desarrollo corporal desde el nacimiento hasta los 63 días de vida parece tener una influencia positiva en el grado de madurez y la productividad al comienzo de su vida reproductiva, además de poder mejorar su supervivencia a corto plazo, pero con posibles efectos negativos en la esperanza de vida a largo plazo. Sin embargo, el desarrollo durante el período de recría (desde los 63 días de vida hasta el comienzo de la carrera reproductiva) parece tener un mayor impacto en la productividad y la esperanza de vida de nuestros futuros conejos reproductores. Las hembras más engrasadas en la primera inseminación tuvieron tamaños de camada más pequeños y un mayor riesgo de ser sacrificadas que las magras (P<0.05). Además, los machos que mostraron una mayor ganancia media diaria durante el período de recría tuvieron un número y porcentaje de eyaculados viables más bajos a lo largo de su vida productiva y mayor riesgo de muerte o sacrificio. Estos resultados podrían indicar que la clave para llevar a cabo un desarrollo adecuado durante el período de recría debería basarse tanto en conseguir que los conejos alcancen un grado de madurez apropiado, tanto fisiológica como reproductivamente, así como evitar un excesivo engrasamiento al inicio de su vida productiva. En los ensayos destinados a evaluar los programas de alimentación durante la recría, el uso de una restricción alimentaria en hembras, y especialmente el uso de dietas fibrosas (de 8.5 a 10 MJ ED/kg MS) permiten un desarrollo corporal gradual y adecuado a la edad de cubrición, con suficientes reservas corporales para afrontar el comienzo de la vida reproductiva y mejorando tanto su rendimiento reproductivo como su vida útil. El éxito de estos programas de alimentación de recría depende del momento y la duración de la aplicación de estos, mejorándose con la aplicación de un cambio a un pienso enriquecido energéticamente alrededor del primer apareamiento. En los machos, se observó que un programa de alimentación durante la recría adecuado debería adaptarse a la capacidad de ingesta de estos y a sus necesidades nutricionales en períodos concretos de la recría, consiguiendo así mejoras en algunos parámetros del semen al inicio de su vida reproductiva. En conclusión, tanto un desarrollo temprano adecuado desde la gestación hasta el comienzo de su vida reproductiva, como el uso de programas adecuados de alimentación durante la recría podrían mejorar significativamente el rendimiento reproductivo futuro, así como la esperanza de vida, tanto en conejos he / [CA] Qualsevol millora en el coneixement tant de la nutrició com del maneig dels futurs conills de criança ha de considerar-se com a prioritari en la producció de conills. No obstant açò, els principals esforços en investigació s'han centrat en aquests animals durant la fase de reproducció pròpiament dita. La hipòtesi inicial d'aquesta tesi va ser que aconseguir un desenvolupament primerenc adequat durant la gestació, lactància, engreix i criança, amb l'ajuda de programes ajustats d'alimentació en alguns d'aquests períodes, milloraria el rendiment reproductiu i la vida útil dels futurs conills reproductors. Per a avaluar aquesta hipòtesi, es van dur a terme dos tipus d'assajos, tant en conills femella de línies maternes, com en mascles de línies seleccionades per a la velocitat de creixement i destinades a la inseminació artificial. El primer d'aquests assajos es va centrar en els diferents patrons de creixement, des del naixement fins al final de la fase de criança, i la seua influència en la carrera reproductiva i la vida útil del conill. En el segon tipus de proves, es va avaluar l'efecte de l'ús de diferents dietes o programes d'alimentació durant el període de criança, en la productivitat a curt i mig termini. Els resultats obtinguts semblen indicar que podríem diferenciar dues fases en el desenvolupament primerenc, d'acord amb els seus efectes sobre el rendiment reproductiu i la vida útil. Mostrar un major desenvolupament corporal des del naixement fins als 63 dies de vida sembla tenir una influència positiva en el grau de maduresa i la productivitat al començament de la seua vida reproductiva, a més de poder millorar la seua supervivència a curt termini, però amb possibles efectes negatius en l'esperança de vida a llarg termini. No obstant açò, el desenvolupament durant el període de sembla tenir un major impacte en la productivitat i l'esperança de vida dels nostres futurs conills reproductors. Les femelles més greixades en la primera inseminació van tenir grandàries de ventrada més xicotets (tant nascuts com deslletats) i un major risc de ser sacrificades que les magres (P<0.05). A més, els mascles que van mostrar un major guany mitjà diari durant el període de criança van tenir un nombre i percentatge d'ejaculats viables més baixos al llarg de la seua vida productiva i major risc de mort o sacrifici. Aquests resultats podrien indicar que la clau per a dur a terme un desenvolupament adequat durant el període de criança hauria de basar-se tant a aconseguir que els conills aconseguisquen un grau de maduresa apropiat, per a afrontar els futurs desafiaments productius, com evitar un excessiu greixatge a l'inici de la seua vida productiva. En els assajos destinats a avaluar els programes d'alimentació durant la criança, l'ús d'una restricció alimentària en femelles, i especialment l'ús de dietes fibroses (de 8.5 a 10 MJ ED/kg MS) permeten un desenvolupament corporal gradual i adequat a l'edat de cobriment, amb suficients reserves corporals per a afrontar el començament de la vida reproductiva i millorant tant el seu rendiment reproductiu com la seua vida útil. L'èxit d'aquests programes d'alimentació de criança depèn del moment i la durada de l'aplicació d'aquests, millorant-se amb l'aplicació d'un canvi a un pinso enriquit energèticament al voltant del primer cobriment. En els mascles, es va observar que un programa d'alimentació durant la criança adequat hauria d'adaptar-se a la capacitat d'ingesta d'aquests i a les seues necessitats nutricionals en períodes concrets de la criança, aconseguint així millores en alguns paràmetres del semen a l'inici de la seua vida reproductiva. En conclusió, tant un desenvolupament primerenc adequat des de la gestació fins al començament de la seua vida reproductiva, com l'ús de programes adequats d'alimentació durant la criança podrien millorar significativament el rendiment reproductiu futur, així com l'esperança / [EN] Any improvement on the knowledge of both nutrition and management of the future rabbit breeders should be considered as crucial. However, main research efforts have been focused on animals during reproduction phase. The initial hypothesis of this thesis was that achieving an adequate early development during young rabbits' gestation, lactation, fattening and rearing, with the help of fitted feeding programmes in some of these periods, would be improved reproductive performance and lifespan of the future breeding rabbits. To evaluate this hypothesis, two types of trials, both in rabbit females from maternal lines selected for litter size at weaning and males from paternal lines selected for growth rate and intended on artificial insemination, were carried out. The first of these trials were mainly focused on the different growth patterns, from birth to the end of the rearing age, and their influence on rabbit's reproductive career and lifespan. In a second type of trials, the effect of the use of different diets or feeding programs during the rearing period in the performance in the short- medium-term was evaluated. The results obtained from the evaluation of the different growth patterns trials seem to indicate that, we could differentiate two parts on the early development according to their effects on reproductive performance and lifespan. To show a greater body development from birth to 63 days of life seems to have a positive influence on the maturity degree and productivity at the beginning of their reproductive life, as well as being able to improve their survival in the short-term, but it could be even negative for long-term lifespan. However, the development during the rearing period (from 63 days of life to the beginning of reproduction career) seems to have a higher impact on the productivity and lifespan of our rabbit breeders. Fatter rabbit females at first insemination had smaller litter sizes (born and weaned) and a higher risk of being culled (+13% per positive change in mm of perirenal fat) than lean ones (P<0.05). Also, rabbit males that showed a greater average daily gain during rearing period had a lower number and percentage of profitable ejaculates through it productive life (-10.1 and -4.9% per each 10 g of daily gain increase, respectively; P<0.05) and higher risk of death or culling (+16.2% per each increase in one standard deviation unit on live weight; P<0.05). These results could indicate that the key to carrying out an adequate development during the rearing period should allow the rabbits to reach an appropriate degree of physiological and reproductive maturity to face the future productive challenges, avoiding an excessive fatness at the beginning of their productive life. The use of a feeding restriction in rabbit females, and especially the use of fibrous diets (from 8.5 to 10 MJ DE/ kg DM) lead a gradual and adequate body development at mating, with enough body reserves to face the beginning of reproduction life and improving their whole reproductive performance and lifespan. The success of these rearing feeding programs depends in the moment and duration of application of them and improved with the application of a nutritional flushing around first mating. In rabbit males, it was observed that an adequate rearing feeding program should adapt it to the male's intake capacity and their nutritional requirements rearing period, especially to their high DP needs at the beginning of rearing period or when their requirements were close to maintenance, to improve some semen parameters at the beginning of their reproduction life. In conclusion, both the adequate early development of future rabbit breeders, from their own gestation to the beginning of their reproductive life, and the use of appropriate rearing feeding programs could significantly improve their future reproductive performance, as well as their life expectancy, both in rabbit females and males. / Martínez Paredes, EM. (2019). Rearing programmes for breeding rabbits. Effect of early development and feeding on future reproductive performance of rabbit females and males [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124969 / Compendio

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.

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 .

Human Immunodeficiency Virus (HIV) infection mostly spreads by the mucosal route: sexual transmission is the dominant mode of transmission, responsible for between 85% and 90% of cases of infection worldwide. These epidemiological data indicate that semen is one of the major sources of HIV-1 transmission. Semen, like other bodily secretions involved in HIV sexual transmission, contains the virus as two forms: cell-free viral particles and cell-associated virus, mostly in infected leukocytes. Although cell-to-cell HIV transmission has been extensively described as more efficient, rapid and resistant to host immune responses, very few studies have investigated the role in vivo of infected leukocytes in virus mucosal transmission. One such study has been recently conducted in our lab, and demonstrated that SIV-infected splenocytes are able to transmit infection to female macaques after vaginal exposure. However, all these studies used immune cells from peripheral blood or lymphoid tissues, such as spleen, and none have investigated the capacity of infected leukocytes in semen to transmit the infection in vivo. Indeed, nature, phenotype and infectivity of HIV associated with semen leukocytes may be different from that of HIV from other sources.Therefore, the objectives of this work are, first, to study of semen leukocytes and their dynamics during SIVmac251 infection in detail, then to investigate seminal factors that may influence semen infectiousness, and finally to test semen leukocyte infectivity in vitro and in vivo, using a model of mucosal exposure in cynomolgus macaques.Macaque semen contains all the target cells for HIV/SIV: CD4+ T cells, macrophages and dendritic cells in lower proportions. Semen CD4+ T cells and macrophages display an activation, differenciation and expression of migration markers profile which is typical of mucosal leucocytes. SIV infection induces significant changes in their phenotype and dynamics. Both cell types can be productively infected and are found in the semen at all stages of infection. These observations suggest that semen CD4+ T cells and macrophages may be able to transmit infection after mucosal exposure.If the role of semen infected leukocytes in HIV/SIV mucosal transmission is confirmed in vivo, this mechanism will be important to consider for further preventive strategies design, like microbicides.

Aquaculture is an essential developing sector for world food production, however the attainment of sexual maturity during commercial on-growing is a major bottleneck to industry expansion. Sexual maturation brings a commercial loss due to reduced growth performance as well as reduced immune function. Furthermore, serious concerns exist over potential genetic interaction with native stocks through broadcast spawning or spawning interaction by escapees. In the north Atlantic region, the Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) are key aquaculture species in which industry expansion is limited by pre-harvest sexual maturation. However, through a species specific combination of modern technologies and refinement in management practices it is possible that this sexual maturation can be controlled and on-growing potential enhanced. Thus the overall aim of this thesis was to conduct novel research that will improve our understanding of the underlying mechanisms that regulate sexual maturation, whilst also advancing the optimisation of technologies for the management of maturation in cod and halibut. In Atlantic cod, owing to the inconsistent inhibition of maturation in commercial conditions, ever increasing intensities of light and in some cases narrow spectrum technologies are being used to try to combat this problem. Firstly, this PhD project investigated the potential welfare impacts of high intensity artificial lighting which have not been studied to date (Chapter 2). The work specifically investigated the effect of traditional metal halide and novel green cathode lighting on the stress response, innate immunity, retina structure, feeding activity and light perception of Atlantic cod. Results indicated that although acute responses to light were observed, there were no clear significant long term effects of any of the lighting treatments on these parameters. Regarding light perception, interestingly even when subjected to high intensity constant lighting (metal halide mean tank intensity: 16.6 watts m-2), cod still demonstrated a day/night rhythm in melatonin release which suggests perception of the overlying ambient photoperiod. The second trial of this PhD project investigated the efficacy of shading of ambient photoperiod in addition to constant lighting to inhibit maturation of cod outdoors (Chapter 3). This aimed at improving the performance of artificial lighting regimes in the open cage system during commercial on-growing by reducing the relative difference between day/night light intensities. The trial was conducted over a one year period where a low and high shade treatment were tested in outdoor tanks. Shading increased the relative night time illumination to 6.6% and 31.3% of daytime levels respectively, compared to <2% in an unshaded set-up. Both shading treatments were effective at suppressing sexual development in cod as confirmed through measurements of gonadosomatic index, histological analysis of gonadal development, oocyte diameter measurements and sex steroid profiles as well as measurements of growth. In addition to research at the applied level in Atlantic cod, this thesis has also extended to the fundamental level and explored one of the potential mechanisms relaying photoperiod signal to the endogenous regulation of sexual maturation in cod, namely the kisspeptin system (Chapter 4). Partial sequences for the signal peptide Kiss2 and its receptor Kissr4 were isolated and described showing similarity to other teleost species such as the medaka, Oryzias latipes and stickleback, Danio rerio. Novel molecular qPCR assays were designed and developed to measure the expression of both genes in male and female cod over a maturation cycle and compared to cod under constant lighting which remained immature. Interestingly, expression patterns of kiss2 and kissr4 did not reveal any clear association with season or photoperiod treatment. However, pituitary expression of gonadotropins (FSH, follicle stimulating hormone; LH, luteinising hormone) did show a differential expression in relation to treatment from early winter approximately 4-6 months after the photoperiod change. These new results are in contradiction with the hypothesis that the kisspeptin system would be involved in the initiation of gametogenesis, as shown in mammals. However, the FSH/LH data defines a window during which time kisspeptin or another GnRH stimulating mechanism must be active, this compels the need further investigation. In Atlantic halibut farming, all-female production removes the concerns of production losses through sexual maturation. Accordingly, this thesis investigated the potential/feasibility of generating monosex populations by FACS (fluorescence activated cell sorting) semen sexing based on cellular DNA content, as proven in terrestrial agriculture. Results however did not show any clear differences between the DNA of sperm in a range of species tested (Atlantic halibut, cod, sea bass, perch) suggesting that this technique may not be applicable in such species. The project also focussed on the production of a population of sex reversed halibut broodstock (neomales) that will generate, in the long term, a basis for traditional monosex population generation in the UK. Two in feed MDHT (17α-methyldihydrotestosterone) treatments were tested with the aim to reduce the use of hormone. Results were very successful with a hormone treatment of 5ppm MDHT generating a 97% phenotypic male population thus suggesting the presence of sex-reversed halibut which can be used for future monosex production. Overall, this work aimed to develop and/or refine potential remediation techniques for sexual maturation in two key commercially important farmed marine fish species, cod and halibut, as well as further our understanding on the regulation of puberty. The knowledge gained from this work provides a means to optimise the techniques employed in the industry and has the potential to increase production and profitability without compromising farmed animal welfare, thus ultimately promoting the sustainable expansion of the Atlantic cod and halibut aquaculture.