Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep[TM], or Cushion Fluid[TM]. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep™, or Cushion Fluid™. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥ 90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

L'objectiu principal d'aquesta tesi és contribuir a un millor coneixement dels mecanismes moleculars implicats en el correcte assoliment de la capacitació "in vitro" (IVC) i posterior reacció acrosòmica "in vitro" (IVAR) dels espermatozoides porcins. Amb aquesta finalitat es va estudiar, per una banda, l'evolució temporal de l'estructura subpoblacional en els espermatozoides porcins durant aquests processos i es va observar canvis espeífics en el percentatge de les diferents subpoblacions mòbils sense que es perdés l'estructura de 4 subpoblacions descrita en l'ejaculat porcí. Per altra banda, la IVC i la IVAR indueixen no sols canvis en la fosforil·lació dels residus tirosina de les proteïnes sinó que s'ha demostrat que la fosforil·lació en els residus serina i treonina també està relacionada amb la IVC i IVAR. Així, s'ha descrit, no tan sols l'aparició de fosforil·lació específica en determinades proteïnes, sinò també la inducció de canvis conformacionals en altres proteïnes i l'aparició de senyal de fosforil·lació en serina i treonina en l'acrosoma de l'espermatozoide relacionada amb IVC i IVAR. En tercer lloc, s'observà un augment de l'activitat mitocondrial durant la IVC i els primers minuts de IVAR. Nogensmenys, en aquest treball es descriu un patró concret d'activació mitocondrial, des de nuclis concrets situats majoritàriament a la part més perifèrica de la peça intermitja de l'espermatozoide cap a la part central d'aquesta. Tanmateix l'actina i la mitofusina2 són dos proteïnes que podrien jugar un paper important en la modulació de la funció mitocondrial en l'espermatozoide porcí. / The first and principal aim of this thesis is to improve our knowledgement in molecular mechanisms and control of "in vitro" capacitation (IVC) and further acrosome reaction (IVAR) in boar spermatozoa. We have studied the structural motile subpopulation, the protein phosphorylation in concrete in tyrosine, serine and threonine residues and finally the mitochondrial activity during IVC and IVAR.Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.Moreover, this study indicates that the changes in protein phosphorylation associated with IVC and subsequent IVAR affected not only pTyr, but also pSer and pThre, and they comprise not only the appearance of specific phosphorylated proteins, like pTyr 32-kDa, pSer-75 kDa and pSer 80-kDa proteins, but also structural changes that induce changes in pI and displacements in the sperm location of the phosphorylated proteins, such as the appearence of specific IVC- and IVAR-linked pSer and pThre signals in the acrosome. Finally, it have been shown that the increase of boar sperm mitochondria activity during IVC and the first minutes of IVAR is instrumental to perform the sperm function changes associated with these processes. Furthermore, the increase in mitochondria activity is originated in concrete nucleation points at the midpiece. Finally, actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondria function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure by itself.

Preservation of cells and tissues by freezing at temperatures below 70°C has led to new possibilities for the storage of germ cells for fertility preservation. During the freezing process problems might occur, the greatest being ice crystallization which can cause membrane destruction and thus cell death. To minimize this risk, solutions that reduce the freezing point can be added to reduce crystallization and increase survival rates. These solutions are called cryoprotectants. The best method for freezing is still not known.The aim of this study was to analyze the effect of various parameters on the survival rate of human semen frozen with liquid nitrogen. The parameters investigated were thawing method (incubator or water bath) and container choice (straw or ampoule). In addition, two different cryoprotectants were tested.The method used was the instruction for preservation with Sperm CryoProtec™ II from Nidacon. In total 16 samples were collected for the first test and 13 samples for the second test. Sperm concentration and motility was measured.There seem to be no significant differences depending on container choice or thawing method leading to the conclusion that the most cost effective method of storage and thawing may be used. A small but significant difference was found in survival after thawing dependent on cryoprotectant p=0.041. However the study sample was limited and further studies might be of value.

Speciation lies at the heart of evolution and the study of reproductive barriers allows a better understanding of the different steps leading to the complete isolation of two species. Pre-mating (behavior tactics, habitat or food divergence, phenotypic divergence and assortative mating) and post-mating, post-zygotic isolation barriers (selection against unfit hybrids) are well studied in numerous species, but little is known about what is happening between insemination and fertilization (post-mating, pre-zygotic isolation barriers). In this study, we chose the well-studied population of pied and collared flycatchers (Ficedula hypoleuca and F. albicollis) of the hybrid zone of Öland, Sweden, to investigate possible patterns of gamete divergence between these two closely related species. We compared sperm morphology between the two species and their hybrids, analyzing traits that are thought to play an important role in the fertilization success of the males. We did not detect any divergence in sperm morphology between the two species, but we report an extreme reduction of sperm production in hybrid males, as well as spermatogenesis dysfunctions and particularly high rates of extra-pair young in the nests of hybrid males.

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep™, or Cushion Fluid™. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥ 90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

This research was carried out in support of fieldwork in the Gulf of Mexico in summers 2004 and 2005 as part of the multidisciplinary Sperm Whale Seismic Study (SWSS). Important aspects of SWSS research include oceanographic habitat characterization and studies of sperm whale foraging and diving patterns. During the SWSS 2005 cruise, acoustic volume backscatter data were collected using a 38 kHz ADCP for comparison with XBT, MODIS ocean color data, and whale dive profiles extrapolated from analysis of towed passive acoustic hydrophone array recordings of whale vocalizations. This unique data set, collected from a cyclonic eddy, was compared with non-upwelling conditions surveyed in the western Gulf and the Mississippi Canyon in summer 2004. My focus was to examine the relationship between acoustic backscatter intensity from the deep scattering layer (DSL; usually 400-600 m deep) and the depths to which whales dived. The results of the study investigate differences in DSL characteristics between divergent zones and non-divergent zones, and examine connections relating to variations in sperm whale dive patterns. The analysis of 38 kHz ADCP data showed that there were significant differences in some characteristics of the main DSL dependent on time of day. There were no significant differences in characteristics of the main DSL between divergent and non-divergent areas or between 2004 and 2005. The comparison of the 38 kHz ADCP and the 70 kHz Simrad echosounder data yielded a relationship of 4 ADCP counts for every 1 dB of Sv. This relationship was a promising start to a potential calibration for the ADCP instrument. Lastly, the analysis of localized sperm whale dive profiles identified three basic dive profiles; Deep (> 800 m), Mid-water dives to DSL depths (500 - 800 m) and Shallow (<500 m). The analysis also showed that whale dive behavior did not change based on time of day or location. It showed that whales are diving above the DSL as well as through and below, however these dives are independent of differences in DSL characteristics.

Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.