Dissertations / Theses on the topic 'Frozen and thawed'

Master of Science
Food Science Institute -- Animal Science & Industry
Daniel Y.C. Fung
In February 2008, the FDA released a draft Compliance Policy Guide (CPG) on Listeria monocytogenes and proposed that ready-to-eat (RTE) foods that do not support the growth of L. monocytogenes may contain up to 100 CFU/g of this pathogen. Frozen foods such as ice cream fall in that category since they are consumed in the frozen state. However, other frozen foods, such as vegetables and seafood that are thawed and served at salad and food bars, may support the growth of Listeria and would not be allowed to contain 100 CFU/g according to the draft CPG. In the current study, growth curves were generated for L. monocytogenes inoculated onto four thawed frozen foods - corn, green peas, crabmeat, and shrimp - stored at 4, 8, 12, and 20ºC. Growth parameters, lag phase duration (LPD), and exponential growth rate (EGR) were determined using a two-phase linear growth model and the Square Root Model. The results demonstrated that L. monocytogenes has a very short LPD on these thawed frozen foods during refrigerated storage and that there would be several orders of magnitude of growth (i.e., more than 1.7 log increase at 4 ºC) of the organism before the product is found to be organoleptically unacceptable. Although it would not be possible to take advantage of any extended lag phase duration caused by freeze injury to the organism, frozen foods containing less than 100 CFU/g of L. monocytogenes that are thawed, or thawed and cooked, and then consumed immediately, should not represent a public health hazard.

Field evaluation of frozen-thawed stallion semen has been limited to tests such as post-thaw motility and morphology that are not only subjective but also evaluate only a small population of cells. Flow cytometry has provided a quick, repeatable, objective method of evaluating a large number of cells, including spermatozoa. Two experiments were designed to first validate the use of several flow cytometric tests on frozen-thawed stallion semen and then determine a model that may best explain variation in fertility. Comparing samples that were live and freeze-killed validated the flow cytometric tests.

In experiment one, six ejaculates were collected from each of three stallions. The semen from each ejaculate was centrifuged and frozen in 0.5 ml polyvinyl chloride straws. Two straws from each ejaculate were thawed and evaluated. Semen was evaluated for post-thaw motility, morphology, mitochondrial activity using Rhodamine 123 (R123), plasma membrane integrity using propidium iodide (PI) and ethidium monoazide (EMA), and chromatin structure using the sperm chromatin structure assay (SCSA). Data was recorded as percentages for all but the SCSA for both experiment one and two. The extent of chromatin denaturation was calculated using the SCSA and the alpha-t population [at = red/(red +green) fluorescence]. From the alpha-t population, statistics were calculated such mean (Xat), standard deviation (SDat), percentage of cells outside (COMPat) the main alpha-t population and the mean green fluorescence (mean green) of the population.

Results from experiment one demonstrated that all flow cytometric tests except EMA were able to distinguish between live and freeze-killed samples (p < 0.0001). Also the stallion accounted for most of the variation in samples when compared to ejaculate and straw within an ejaculate. Therefore two straws could be chosen at random from a stallion and evaluated in experiment two.

In experiment two, twenty-nine stallions were evaluated using the same tests as experiment one excluding EMA. Fertility data was obtained from the 1998 or 1999 breeding season. Multiple linear regression was used to determine the best-fit model to predict overall pregnancy rate. SCSA and R123-PI assays accounted for the largest amount of variation in fertility (R² = 0.65, p < 0.0004). Within SCSA and the R123/PI assays Xat and PI staining had the highest contribution to this variation in fertility (R² = 0.11, R² = 0.47) respectively. The best-fit model for predicting fertility included the assay combination listed above and the interactions between SDat and mean green staining as well as R123 and mean green staining. Post-thaw motility and morphology did not account for significant variation in fertility (p = 0.22, p = 0.46) respectively.

Based on this project post-thaw motility and morphology are poor predictors of fertility in frozen-thawed stallion semen. However, through the addition of SCSA and R123-PI to the routine evaluation of frozen-thawed stallion semen time and money may be saved in advance by identifying those stallions with poor post-thaw fertility.
Master of Science

Determination of an extender protocol which will enhance the viability of frozenthawed bovine spermatozoa will allow producers to obtain higher conception rates due to the increased survival rate of the spermatozoa. Ejaculates of six Brangus bulls (age=18 months) were evaluated for spermatozoal motility, acrosomal integrity, and morphological characteristics (collectively called spermatozoal viability) in two experiments to test our hypotheses that (1) the treatment combination of a 4 hr cooling duration and a 2 hr equilibration with glycerol will result in optimum spermatozoal characteristics after freezing and thawing and (2) rank of three selected extenders relative to their effects on spermatozoal viability after freezing and thawing will be egg yolk-citrate (EC), egg yolk-tris (IMV), and skim milk (milk). In experiment 1, an ejaculate from each bull was partially extended and cooled to 4 ºC for either 2 or 4 hr and then allowed to equilibrate with the glycerolated extender for 2, 4, or 6 hr. Spermatozoal viability was assessed at 0, 3, 6, and 9 hr after thawing. In experiment 1, 4 hr of cooling resulted in a higher percentage of motile spermatozoa than did 2 hr of cooling. The 2 hr equilibration with glycerol yielded lower percentages of motile spermatozoa, acrosomal integrity, and morphologically normal spermatozoa than 4 and 6 hr equilibration durations with glycerol. In experiment 2, we observed a decrease in spermatozoal viability for all three extenders upon freezing and thawing. Viability of frozen-thawed spermatozoa extended in the milk was reduced for all incubation durations, and the IMV extender had a higher percentage of motile spermatozoa than the EC extender at 6 hr of incubation. A higher percentage of intact acrosomes was observed with the IMV extender; however, the EC extender had a higher percentage of morphologically normal spermatozoa than the IMV extender. Our results indicate that at cooling duration of 4 hr and a 4 hr equilibration with glycerol provide the highest level of spermatozoal viability post-thaw of the treatments evaluated and that the IMV extender enhances the percentage of spermatozoa with an intact acrosome for frozenthawed spermatozoa over the EC and skim milk extenders.

The aim of the current study was to determine if frozen-thawed bull semen can be treated with the acidic buffer MES (2-[N- morpholino] ethanesulfonic acid) without any detrimental effect on the motility, plasma membrane, acrosomal membrane and longevity of sperm. Frozen bull semen was obtained from a local co-operative. The semen was frozen in 0.25 mL French straws at a concentration of 80 x 106 sperm cells per millilitre. Semen of two different batches from ten bulls of four different breeds was used in this study. Three frozen semen straws of each batch were thawed at 38° C for 25 seconds. The thawed semen was pooled and then split into two aliquots. The one aliquot was used as control, whilst the other was exposed to MES treatment. The motility, plasma membrane integrity, acrosomal membrane integrity and longevity of sperm were evaluated. The effect of MES on motility was minimal as only the percentage of aberrantly motile sperm increased two hours after treatment. Although no effect on the plasma membranes were observed, it can be assumed that some damage did occur due to the fact that the acrosomal membranes were affected significantly. No significant effect was found for longevity of sperm between the control and treated samples, but a significant effect was found for both the control and treated samples over time. Although the detrimental effects caused by MES treatment would render some sperm unable to fertilise an oocyte, it is likely that a sufficient portion of sperm would survive the treatment. It is probable that this treatment would also be effective in frozen-thawed buffalo semen. The following step would be to treat semen of footand-mouth disease positive bulls with MES to establish if treatment with MES will be effective in inactivating foot-and-mouth disease virus in semen of infected bulls. Copyright
Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008.
Production Animal Studies
unrestricted

Se utilizarán eyaculados procedentes de machos de la línea R (línea de conejos seleccionada por velocidad de crecimiento durante el periodo de engorde)alojados en diferentes centros de inseminación artificial. Una vez recuperados los eyaculados se procederá a su valoración y una muestra de todos ellos será crioconservada. La calidad seminal será de nuevo valorada tras el proceso de congelación. Junto con los anàlisis seminales se utilizarán los datos de crecimiento y pedigree de los machos y de todos los animales de la línea R desde su fundación para estimar por un lado los parámetros genéticos de las variables relacionadas con la producción y calidad de dosis seminales en fresco y tras un proceso de crioconservación y la correlación genética existente entre las variables seminales anteriormente citadas y la velocidad de crecimiento. A su vez se estimará mediante un modelo recursivo la relación entre las variables seminales en fresco y tras la descongelación.
The general aim of this thesis was to study the genetic determinism for some traits related to artificial insemination (AI) dose production of fresh and frozen-thawed semen, in order to explore the interest and limitation of different strategies for their genetic improvement in a paternal line of rabbits selected for growth rate during the fattening period (28-63 days). In chapter 1, genetic parameters of sperm production traits are estimated as well as the genetic relationship with daily gain (DG). The heritabilities (h2) of the semen traits were 0.13±0.05, 0.08±0.04 and 0.07±0.03 for ejaculate volume (V), sperm concentration (CN) and sperm production (PROD) per ejaculate, respectively. A favourable and moderate genetic correlation was observed between V and DG (0.36±0.34). From this chapter it may be concluded that if a seminal trait is to be included as a selection objective, a useful one could be sperm production, as it is a trait in which both volume and concentration are included. Moreover, there is currently no evidence to suggest that selection for DG in rabbits will affect sperm production adversely. The aim of chapter 2 was to explore the genetic determinism of some sperm quality traits and their genetic relation with the selection criteria of the paternal rabbit line. The heritabilities (h2) of semen quality traits commonly evaluated in a classic spermiogram were 0.18, 0.19 and 0.12 for NAR (%, percentage of sperm with intact acrosome), ANR (%, percentage of sperm abnormalities) and MOT (%, percentage of total motile sperm cells) respectively. We also estimated the h2 of some motion CASA parameters 0.09, 0.11, 0.10, 0.11, 0.11 and 0.11 for VAP (µm/s; average path velocity), VSL (µm/s; straight-line velocity), VCL (µm/s; curvilinear velocity), LIN (%, linearity index), ALH (µm; amplitude of the lateral head displacement), STR (%, straightness). Genetic correlations between DG and semen traits showed a high HPD95% (interval of highest density of 95%). However there is some consistent evidence of the negativity of the genetic correlations of DG with NAR and MOT (-0.40 and -0.53, respectively). Chapter 3 aims to determine the repeatability and heritability of sperm head characteristics: width (W, ¿m), area (A, ¿m2),length (L, ¿m) and perimeter (P, ¿m), and explore the relationships between them and with the selection objective (DG). The results obtained showed that sperm head dimensions are heritable (ranged between 0.2 and 0.29). The genetic correlations between sperm traits were always high and positive (between 0.72 and 0.90), with the exception of L-W genetic correlation, which was moderate. Regarding the genetic correlations between DG and sperm head characteristics, the resulting means ranged from -0.09 for L-DG to -0.43 for W-DG, showing consistent evidence of the negativity of the genetic correlations. The environmental and male effects that could have an influence on sperm freezability are studied in Chapter 4. Six different traits were evaluated: sperm concentration (CONC, 106spermatozoa/mL), acrosome integrity in fresh (NAR, %) and frozen-thawed semen (Nar-FT, %), sperm motility in fresh (MOT, %) and frozen-thawed semen (Mot-FT, %) and the percentage of viable sperm in frozen-thawed semen (Live-FT, %). In addition, two synthetic traits were computed: the relative reduction of acrosome integrity (Rnar, %) and relative reduction of motility (Rmot, %) after the freezing-thawing process. A multiple-trait recursive model was used to analyse the relationships between the semen traits considered. For the fixed effects studied, the season had the highest impact on post-thaw semen characteristics. Results of the analysis of recursive coefficients showed that fresh semen concentration and motility influence the future freezability of the semen. All traits studied presented moderate repeatabilities, ranging from 0.11 to 0.38. These results provide conclusive evidence that sperm freezability in rabbits could be heritable. Regarding male correlations, there were large positive male correlations between fresh traits (rm=0.77-0.57), as well as between direct frozen-thawed traits (rm=0.72-1). Male effects on fresh and direct frozen-thawed traits were generally positively correlated. This correlation was moderate to high for MOT with all frozen-thawed traits (rm=0.41-0.74) and for Mot-FT and all fresh traits (rm=0.5-0.74); these results suggest that these traits could be genetically related. The final chapter of this thesis focused on estimating the heritability of semen freezability traits and estimating the genetic correlation between frozen-thawed sperm traits and the growth rate in a paternal rabbit line. Estimated heritabilities showed that frozen-thawed semen traits are heritable (ranged between 0.08 and 0.15). In the case of Live-FT, the estimated heritability is the highest and suggests the possibility of effective selection. After the study of genetic correlations, it seems that DG was negatively correlated with sperm freezability, but due to the high HPD95% no further conclusions could be drawn. More data should be included in order to obtain better accuracy for the estimates of these genetic correlations. If the results obtained in the present study were confirmed, it would imply that selection for DG could alter sperm cell membranes or seminal plasma composition, both components related to sperm cryoresistance.
Lavara García, R. (2013). Genetics of fresh and frozen-thawed semen traits and their relationship with growth rate in rabbits [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31657
TESIS

The ability to accurately evaluate the quality and estimate the fertilizing potential of dog semen has increased in importance as a result of the more widespread use of artificial insemination but remains challenging. The actual conception rate (CR) of a particular male of this polytocous species may provide useful information particularly in a competitive setting of a multi-sire mating or insemination. A crucial part in establishing the actual CR of a male is played by the fecundity of the female-which may ovulate from multi-ovular follicles-as well as breeding timing, breeding technique, and number of spermatozoa inseminated. This thesis determined, in a multi-sire insemination trial using DNA analysis and paternity testing, that the optimal day for surgical insemination using frozen-thawed semen was six days after the rise of the plasma progesterone concentration to between 6 and 9 nmol/L. Concurrently, the frozen-thawed semen used in the insemination trial was evaluated by means of conventional and modern semen evaluation methods, one of which, namely the Merocyanine 540 staining method, was newly validated on fresh dog sperm. Conventional assessment of sperm quality variables included individual progressive motility, viability and morphology using eosin-nigrosin staining. More modern semen evaluation procedures such as epifluorescence microscopy and flow cytometry were used to assess viability (Ethidium Homodimer, Yo-Pro 1), capacitation status (Anti-phosphotyrosine Clone 4G10), membrane destabilization (Merocyanine M540), acrosomal status (FITC-PNA), presence of progesterone receptors (P-BSA-FITC), motility parameters assessed using CASA, as well as the defragmentation index of sperm chromatin (SCSA). Males were ranked according to their CR which was then correlated to 40 sperm quality variables. Two sperm subpopulations, namely the percentage of live sperm which show signs of membrane destabilization (negatively), and the ability of sperm to maintain their viability (positively), did correlate or tended to correlate to in vivo fertility of the males. Another aspect of this thesis estimated the overall probability of a bitch having more than one conceptus derived from a smaller number of follicles, by retrospectively evaluating data of fertility trials as well as collecting data from private practice and welfare organization, thus establishing that the number of corpora lutea of a bitch may be used as a measurement for her fertility, despite the occurrence of multi-ovular follicles in the bitch. The current thesis assessed different aspects of male and female fertility in the domestic dog which, used in conjunction, may increase the ability to accurately estimate the fertilizing potential of frozen-thawed dog semen.
Thesis (PhD)--University of Pretoria, 2017.
Production Animal Studies
PhD
Unrestricted

The aim of the study was to determine the fatty acid composition, colour stability and lipid oxidation of fresh mince produced from fallow deer and to evaluate the effect of frozen storage duration on the retail display shelf life of the mince. A total of 31 fallow deer carcasses were used in the study. After cooling for 24hrs, the carcasses were deboned, external fat from the fore and hindquarter muscles removed and individually vacuum packed. For the first trial, seven fallow deer carcasses were used. Meat from the hind and fore-quarters of each carcass was divided into two equal batches per animal. One batch was minced (through a 5 mm die) and packed into oxygen permeable overwraps and refrigerated at 4°C for a period of eight days under retail display conditions. The second batch was vacuum packed and frozen at -20°C for 2 months at the end of which mince was also produced and monitored over an eight-day period under the same conditions that were used for the fresh mince. Colour, pH, lipid and myoglobin stability was determined. Proximate and fatty acid composition was also determined. No differences (P>0.05) were noted between proximate composition of fresh and frozen/thawed minced meat. The lipid content of fallow deer was 2.4 percent (±0.04). Total n3 fatty acids differed (P<0.05) between treatments and decreased with increased storage and display day. There were significant (P<0.05) treatment and time interactions on all measured colour parameters, TBARS and myoglobin forms. Fresh mince was lighter and had higher redness (a*) and yellowness (b*) values than mince from two months frozen stored meat. Hue angle for fresh mince remained stable throughout display whereas it increased for frozen/thawed mince. Fresh mince had lower TBARS values than frozen/thawed mince. Minced meat produced from frozen/thawed deer meat had higher surface met-myoglobin and total met-myoglobin percentages. Surface and total oxy-myoglobin percentage was higher in fresh mince. The first trial clearly showed colour and lipid stability differences between fresh mince and mince from frozen/thawed meat. It also showed that fresh mince has a longer retail display life than mince produced from frozen/thawed meat (six days and four days, respectively). In the second trial, the effects of frozen storage duration on colour and lipid stability were investigated. Twenty-four fallow deer were used. Twelve were harvested in June (6male 6female) and the other twelve in August (6 male 6female) of the same year.Twenty four hours after harvesting, the fore and hindquarter muscles of the carcasses were deboned, vacuum packed and kept at -20°C until October (i.e. 2months and 4months frozen storage period). Upon thawing, the meat was processed into mince following the same procedure used for the first trialand displayed for a fiveday period under retail display conditions. Frozen duration and gender had no effect (P>0.05) on the proximate composition of fallow deer meat. The total amount of saturated fatty acids (SFA) increased and total amount of poly unsaturated fatty acids (PUFA) decreased as frozen duration and display day increased (P<0.05). Frozen duration affected (P<0.01) lipid oxidation and percentage oxy-myoglobin. Mince pH and all colour parameters (L*, a*, b*,hue and chroma) differed (P<0.05) between treatments on day zero and three. Display day was a significant factor (P<0.05) on all measured parameters. By day three all parameters except pH showed signs of extended oxidation and discolouration as evidenced by reduced redness, decreased colour intensity and high TBARS values. This study showed that prolonged frozen storage negatively affects the colour and lipid stability of meat and increases oxidation of PUFAs during frozen storage. However, the study also suggests that although frozen/thawed meat has a shorter retail display shelf life; the proximate composition of the meat remains unchanged.

Abstract Fertility declines with advancing age and the number of couples seeking infertility treatment at an older age is constantly increasing. A top quality embryo is believed to have the highest potential for implantation and development into a child. A better understanding of the relative importance of patient and treatment characteristics and of embryo quality could help to optimise the existing therapeutic schemes and the safety of in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI). In this work, databases of five Finnish infertility clinics were studied retrospectively. Data on treatments performed in the years 1994–2005 were collected. A total of 19,000 treatment cycles were analysed. Special attention was paid to the relative significance of the transfer of top quality embryos with regards to pregnancy, miscarriage, live birth and cost of treatment in the general IVF/ICSI patient population and in groups with expected poor outcome. The results showed that the transfer of a top quality embryo is associated with a better chance of pregnancy and live birth. However, it does not diminish the probability of miscarriage. Both low and high BMI increase the miscarriage rate. Advancing age and a positive history of previous miscarriages are also associated with a higher probability of miscarriage. In addition, the need for hormonal substitution in cases of frozen-embryo transfer is a risk factor of miscarriage, probably because of suboptimal endometrial function. Since the transfer of several embryos leads to multiple pregnancies, which are associated with a high risk of maternal and fetal complications, elective single embryo transfer (eSET) of a top quality embryo allows all additional good quality embryos to be frozen and transferred later in frozen-thawed embryo transfer cycles. The present work demonstrates that eSET is a safe treatment strategy at least until the age of 40. However, it might not be performed in women with fewer than four collected oocytes, since the prognosis might remain poor even if the response is improved in a following cycle. When eSET is applied routinely and on a large scale, it diminishes treatment costs while increasing the number of deliveries occurring at term, making IVF/ICSI at the same time safer and more affordable even to patients without access to reimbursed IVF treatment.

A study was conducted on the thaw consolidation behaviour of fine grained permafrost soils, which are commonly found in the Mackenzie Valley, Canada. In 1971, Morgenstern and Nixon proposed a thaw consolidation theory. A laboratory study has been carried out with a focus on the consolidation behaviour of thawing fine grained permafrost soils for a case where the thawing soil consolidates under its own weight. Numerical modelling was carried out to predict the temperature behaviour and moisture migration observed in the laboratory. The design of the laboratory apparatus is discussed and the results are evaluated in comparison with the numerical and theoretical predictions. The finite element software are proved useful in predicting the soil thermal and hydraulic behaviour. Although the laboratory results and that from the thaw consolidation theory developed by Morgenstern and Nixon (1971) cannot be compared directly due to many uncertainties, the laboratory results are in reasonable agreement.

Soil is the most widely used material in the construction of various civil infrastructure. Various types of soils are extensively used in its natural or compacted form in the construction of dams, canals, road and railway subgrades, and waste containment structures such as soil covers and liners. These infrastructure and foundation soils are exposed to the influence of environmental factors. In the permafrost and seasonally frozen regions, soils can be in different states (e.g., saturated or unsaturated, frozen or thawed, or combinations of them) due to the variations in moisture content and temperature. The soil-water characteristic curve (SWCC), which is the relationship between soil water content and suction, is used in the interpretation and prediction of unsaturated soils behavior. Similarly, the soil-freezing characteristic curve (SFCC), which is the relationship between unfrozen water content and subzero temperature, is used in the prediction and interpretation of frozen soils behavior. In this thesis, the SWCC and SFCC of two Canadian soils (i.e. Toronto silty clay (TSC) and Toronto lean clay (TLC)) were extensively investigated for better understanding the fundamental relationship between SWCC and SFCC. The soil resilient modulus (MR) is a key material property used in the rational design of pavements. Experimental investigations were undertaken to determine the MR of five Canadian soils (i.e., TSC, TLC, Kincardine lean clay (KLC), Ottawa Leda clay (OLC), and Indian Head till (IHT)), considering the influence of moisture and temperature, with the aid of an advanced triaxial testing equipment. Two simple models were proposed for estimating the MR of frozen soils, in this thesis. In addition, an artificial neural network (ANN) model was developed for estimating the MR of the five Canadian soils considering various influencing factors. The conclusions from the various studies in this thesis are succinctly summarized below. (1) Four expressions (i.e. power relationship, exponential relationship, van Genuchten equation, and Fredlund and Xing equation) that are widely used for representing the SFCC were selected for providing comparisons between the measured and fitted SFCCs for different soils. The results suggest that the exponential relationship and van Genuchten equation are suitable for sandy soils. The power relationship reasonably fits the SFCC for soils with different particle sizes, but not for saline silts. The Fredlund and Xing equation is flexible and provides good fits for all the soils. (2) The SFCC and SWCC of TSC and TLC were experimentally determined, analyzed, and compared. Many factors influence the reliable measurement of SFCC, which include sensors’ resolution and stability, sensor calibration for each soil, and thermodynamic equilibrium condition. The hysteresis of SFCC for the two soils is mainly attributed to the supercooling of pore water. The quantitative dissimilarity in the measured SFCC and SWCC may be attributed to specimen structure variations during compaction and saturation, and during freezing / thawing processes, and cracks formation due to sensors insertion. In addition, some fundamental differences may exist between the drying / wetting and freezing / thawing processes, resulting in dissimilarity. (3) Two novel models were proposed for the estimation of MR of frozen soils. The semi-empirical model extends the mechanics of unsaturated soils and employs SFCC for prediction. Several coarse- and fine-grained saturated soils were used to validate this model. The empirical hyperbolic model was proposed considering that the frozen MR versus subzero temperature relationship resembles hyperbola. This model was validated on coarse- and fine-grained soils under saturated / unsaturated conditions. The hyperbolic model has wider application since it can be used for both saturated and unsaturated frozen soils. Both the models are simple and promising. (4) The MR of five Canadian soils subjected to wetting and freezing was determined by using the GDS ELDyn triaxial testing system. A freezing system was established for controlling the desired testing temperatures within the soil specimens. The results suggest: (i) The effect of subzero temperature on the MR is significant. (ii) For TLC, KLC, OLC, and IHT, the frozen MR versus subzero temperature relationship of the saturated specimen typically has steeper slope than specimen at the optimum water content, for the temperature range from 0 to -5 °C. (iii) The effect of stress levels on the frozen MR depends on soil type, water content, and subzero temperature. Lastly, (iv) Loading frequency does not show a significant influence on the frozen MR. (5) The MR of the five Canadian soils was determined considering wetting and freeze-thaw (F-T) conditions. The results suggest: (i) The F-T cycles result in weak soil structure due to reduction in suction, particles movement, loss of cohesion, and formation of cracks / channels. (ii) The critical numbers of F-T cycles were determined as 1, 1, 2, and 1 for TLC, KLC, OLC, and IHT at the optimum water content, respectively. (iii) The percentage of reduction in MR after the critical number of F-T cycles was strongly related to the plasticity index for specimens tested at the optimum water content. (iv) The wetting process results in the decrease in suction and enlargement of soil pores. Consequently, relatively low MR values were measured at high water contents, and the effect of F-T cycles becomes insignificant. Finally, (v) The effect of stress levels on the MR was dependent on the initial water content of the specimen and soil type.

This project concerns the effect of freeze-thaw cycles on the pore-scale structure of nonaqueous phase liquid (NAPL) contaminants in water-saturated porous media. This problem is of critical importance to the entrapment of such contaminants in cold temperate, polar and high altitude regions, and has not been examined in the literature to date. This research work is conducted in three stages: (i) two-dimensional nondestructive visualisation of residual light non-aqueous phase liquid (LNAPL), and dense non-aqueous phase liquid (DNAPL), in porous media subject to successive freeze-thaw cycles; (ii) three-dimensional experiments on LNAPL in porous media subject to freeze-thaw, with quantification of phase volumes by X-ray micro-computed tomography (micro-CT); and (iii) the explanation of results by several pore scale mathematical and conceptual models. The two-dimensional cell experiments (using a monolayer of 0.5 mm diameter glass beads held between two glass sheets), and three-dimensional X-ray micro-CT experiments reveal a substantial mobilisation and rupture of ganglia during successive freeze-thaw cycles; this includes the detachment of smaller ganglia from larger ganglia and the mobilisation of NAPL in the direction of freezing front. The experiments also reveal significant shedding of numerous single/sub-singlet ganglia along narrow pore corridors, their entrapment in growing polycrystalline ice, and the coalescence of such small ganglia during thawing to form larger singlets. These changes were more predominant where the freezing commenced. The results of the experimental studies were interpreted by developing several mathematical and conceptual models, including freezing-induced pressure model, Darcy's law model, multipore ganglia model (rupture coefficient) and ice-snap off model.

碩士
國立屏東科技大學
動物科學與畜產系所
99
Sperm cryopreservation is one of the basic technologies in reproduction biology. However, the quality of frozen-thawed goat semen is still not as good as that of the fresh semen. The types and components of cryoprotectants might be the key points for improving the goat semen quality after frozen and thawed. Therefore, the objective of this study is to investigate the effects of concentration of low-density lipoprotein (LDL), amino acid and pH value on frozen-thawed semen quality in Alpine goats. In the following research was to establish the extracting method of LDL from hen’s egg yolk. The results showed that the recovery rate of our extraction method was 60.37%. In the study 1, various concentrations of LDL (between 0 and 20%, w/v) were tested in freezing extenders for Alpine goat semen cryopreservation. The results showed that, the motility (72.65%), survival rate (79.74%), and mitochondrial potential (72.77%) of frozen-thawed Alpine goat semen cryopreserved in extender containing 4% LDL were significantly better than those of other treatment groups and the control group (P &lt; 0.05). In study 2, the experiment 1 was to evaluate the effects of pH value of extender on the quality of frozen-thawed Alpine goat semen. The results showsed that quality of frozen-thawed Alpine goat semen in 4% LDL extender was significantly improved when its pH value was adjusted to 6.4 when compare to that of pH 5.7 and 7.0 (P &lt;0.05). In the experiment 2 of study 2, the effects of commercial Roswell Park Memorial Institute-1640 (RPMI-1640) amino acids on the quality of frozen-thawed Alpine goat semen was evaluated. Various concentrations of RPMI-1640 (between 10 and 50%, v/v) in freezing extenders were tested. The results showed that the motility (73.29%), survival rate (77.27%), and mitochondrial potential (68.27%) of frozen-thawed Alpine goat semen were significantly better in the extender containing 40% 1X RPMI-1640 than other treatment groups and the control group (P &lt; 0.05). In conclusion, the traditional role of the egg yolk using as a cryoprotectant in Alpine goat semen cryopreservation extender could be substituted by LDL. In addition, the cryopreservation extender containing 4% LDL and 40% RPMI-1640 and its pH value was adjusted to 6.4, can achieve the best cryopreservation effect for the Alpine goat semen.

碩士
國立屏東科技大學
動物科學與畜產系所
105
Cryopreservation process produces significant changes in mammalian spermatozoa with the subsequent decrease in its fertility potential. Different factors are involved in the alteration of sperm during the cryopreservation: the cryoprotectant used and chilling, freezing and thawed processes employed. Therefore, a large number of extenders have been studied in order to preserve the canine semen quality during the cryopreservation. This study was investigated to explore the effect of glycerol (G), low density lipoprotein (LDL) and trehalose concentration on post-thawing canine semen quality. Semen were collected from three mature Maltese canine (2 and 5 years of age)；The aim of this study is to evaluate the semen quality and is divided into motility (%), progressive (%), viability (%), acrosome integrity (%), mitochondrial integrity (%), and DNA integrity. Results showed that the final glycerol concentration of 5% of the cryoprotectant is significantly higher (P <0.05) than other extender is 3%, 4%, 6% and 7% glycerol concentration in 11% lactose with 20% egg yolk basic extender. When the extender added 11% LDL, canine semen quality (P < 0.05) is significant higher than 10%, 12%, 13% and control group (20% egg yolk) in 11% lactose with 5% glycerol-based extender. Based on the 11% LDL with 5% glycerol based extender, the final optimal trehalose concentration of canine semen cryoprotectant is 350 mM which is better than 300 mM and the control group (11% lactose). In conclusion, the best concentration of glycerol, LDL and trehalose for freezing canine semen are 5 %, 11% and 350 mM. Keywords: Canine, Cryoprotectation, low density lipoprotein, Glycerol, Semen quality, Trehalose

Artificial insemination (AI) with sex-sorted bottlenose dolphin spermatozoa provides female calves for obtaining more cohesive social groups and optimum genetic management of captive populations. However, distance of animals to the sorting facility represents a limit to the procedure. Although one bottlenose dolphin calf has been born using spermatozoa from frozen-thawed, sorted and recryopreserved spermatozoa, critical evaluation of the steps involved in this process is required to maximize its efficiency for future AIs and expansion of the technology to other species. Two experiments were designed to determine the efficiency of the sorting process and the quality of frozen-thawed bottlenose dolphin spermatozoa during sorting and recryopreservation. In experiment 1, the effect of two washing media (with and without 4 percent egg yolk, v/v) following density gradient centrifugation (DGC) on sperm recovery rate and in vitro characteristics of cryopreserved spermatozoa was examined. In experiment 2, cryopreserved semen was used to compare the effects of two recryopreservation methods (conventional straw freezing and directional freezing) on in vitro sperm characteristics of control (non-sorted) and sorted spermatozoa. Egg yolk supplementation of the washing medium in experiment 1 did not influence (P > 0.05) the sperm recovery rate, however, sperm motility parameters and viability were improved (P < 0.05). For Experiment 2, motility parameters and viability were influenced by stage of sex-sorting process, sperm type (non-sorted and sorted) and freezing method (P < 0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P < 0.05) motility parameters over the 24 h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of nonsorted spermatozoa, as assessed by the SCSA, remained unchanged throughout the process. However, a possible interaction between Hoechst 33342 and acridine orange was observed in sorted samples. After recryopreservation, viability of sorted spermatozoa was higher (P < 0.05) than that of non-sorted spermatozoa across all time points. The percentages of viable spermatozoa determined by light (eosin-nigrosin) and fluorescence microscopy (propidium iodide) techniques were correlated (R^2=0.79, P < 0.001). Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

碩士
國立臺灣大學
臨床醫學研究所
90
Summary Introduction Upon release from the male, mammalian sperm are nonfertilizing, but become functionally competent cells with the capacity to fertilize physiologically both in the female reproductive tract and / or under appropriate conditions in vitro, a process so-called “capacitation”. Capacitation involves both sperm surface and intracellular changes. Certain requirements for the modulation of capacitation have been identified : Fertilization promoting peptide ( FPP; pGlu-Glu-ProNH2 ), a tripeptide produced by the prostate gland and secreted into seminal plasma in several mammals, is a potential modulator of capacitation in vivo. At ejaculation, nonfertilizing mammalian sperm come into contact with FPP, which elicits capacitation — dependant responses, i.e. stimulating capacitation in uncapacitated sperm and then inhibiting spontaneous acrosome reaction in capacitated cells. FPP thus initiates and maintains fertilizing potential of sperm until they contact an oocyte in the female reproductive tract. Adenosine, another compound found in mammalian seminal plasma, elicits similar capacitation — dependant responses in sperm cells to FPP. The combination of FPP plus adenosine, whether mixed at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. This suggests that the two molecules act via separate external receptors and modulate a common signal transduction pathway. Adequate sperm motility is known to be an essential prerequisite for successful fertilization. Pentoxifylline, clinically used to enhance motility of sperm from infertile human subjects, is a phosphodiesterase inhibitor leading to an increase in intracellular cAMP level. In hamsters, pentoxifylline has been shown to induce capacitation, hyperactivation and acrosome reaction, and also to improve fertilization rates in vitro. The application of pentoxifylline could contributes to enhancement of post — thawed sperm motility and selection of viable sperm in intracytoplasmic sperm injection. Both FPP and adenosine may act as a first messenger, via adenylate cyclase ( AC ) / cAMP signaling pathway, to modulate the fertilizing ability of mammalian sperm. Cyclic AMP, therefore, plays an essential role in the mechanisms of capacitation and acrosome reaction, which are modulated by either external signal molecules such as FPP and adenosine, or by artificial sperm stimulants such as pentoxifylline. Herein, we try to evaluate the effects of FPP, adenosine and pentoxifylline on the fertilizing ability and motility characteristics of frozen — thawed human sperm. Materials and Methods Semen Sample Semen obtained by masturbation was provided by volunteer donors. After liquefaction and basic seminalysis, the donated semen sample was stored by cryopreservation. On the day of examination, the frozen semen sample was thawed, and the post — thawed sperm suspension was then divided into 2 to 4 aliquotes, one of which was used as the control. Just after thawing, the other aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. Thereafter, we assessed the effects of these three reagents on frozen — thawed human sperm at 1 hour and 4 hours of incubation. Part I. Chlortetracycline ( CTC ) Fluorescence Assessment We collected 16 semen samples, all which were divided into 4 aliquotes of sperm suspension after thawing. One of the 4 aliquotes was used as the control, and the other aliquots were added with reagents to make 100nM FPP - treated, 10μM adenosine - treated, and 1 mg/mL pentoxyfilline - treated sperm suspensions respectively. Most cytological techniques uased to determine the effects of treatment on sperm capacitation only identify acrosome — intact cells and acrosome — reacted cells。With CTC, the acrosome — intact category can be subdivided into two on the basis of apparent differences in their functional state, i.e. uncapacitated and capacitated: F, with uniform fluorescence over the entire head, characteristic of uncapicitated, acrosome-intact cells B, with a fluorescence - free band in the post-acrosomal region, characteristic of capicitated, acrosome - intact cells AR, with dull or absent fluorescence over the sperm head, characteristic of capicitated, acrosome - reacted cells In addition to CTC , we use Hoechst 33258 to assess the live / dead status of the sperm. In each sample, 200 Hoechst 33258 negative cells were assessed for CTC staining patterns under Nikon’s eclipse E600 microscope equipped with phase — contrast and epifluorescent optics. Part II. Computer Aided Sperm Analyzer ( CASA ) Depending on the post — thawed semen qualities, we divided the sperm suspensions into 2 to 4 aliquotes. One aliquote was used as the control, and the other 1 to 3 aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. There were 27 cases with FPP — treated aliquotes, 16 cases with adenosine — treated aliquotes, and 23 cases with pentoxyfilline — treated aliquots in this experiment. Using CASA, we assessed the following motion parameters in each sample: Straight — line velocity ( VSL )：the straight — line distance between the beginning and the end of the track divided by the time elapsed Curvilinear velocity ( VCL )：the total distance between each position of the cell center of brightness ( CB ) for a given cell during the acquisition, divided by the time elapsed Average path velocity ( VAP )：the smoothed average position of the CB and gives an average cell path velocity Amplitude of lateral head displacement ( ALH )：the mean width of the head oscillation as the cell swims Beat cross frequency ( BCF )：the frequency with which the cell track crosses the cell path in either direction Straightness ( STR )：the departure of the cell path from a straight line Linearity ( LIN )：the departure of the cell track from a straight line Statistic Analyses All statistic analyses were carried out by paired t — test. Percentage data of the CTC results were subjected to arc — sine transformation before statistic analyses. Data were expressed as mean ± SEM and P≦0.05 was considered to be statistically significant. Results Part I. Chlortetracycline ( CTC ) Fluorescence Assessment CTC fluorescence assessment showed no significant differences between control group and FPP — treated, adenosine — treated, pentoxifylline — treated groups. All FPP, adenosine, and pentoxifylline failed to improve the fertilizing ability of frozen — thawed human sperm. Part II. Computer Aided Sperm Analyzer ( CASA ) At 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated groups were significantly greater than that of control group. In addition, pentoxifylline significantly lessened LIN up to 4 hours of incubation. Although FPP did not speed the frozen — thawed human sperm, it did modify the motility characteristics. At 1 hour of incubation, the BCF of FPP — treated group was significantly smaller than that of control group. At 4 hours of incubation, FPP made the STR and LIN much less than that of the control group. Pentoxifylline did not affect the motility rate of the frozen — thawed human sperm. However, the motility rates of both the FPP — treated and adenosine — treated groups were unexpectedly lower than that of the control group at 1 hour of incubation. Discussion CTC fluorescence assessment showed that none of FPP, adenosine and pentoxifylline have significant effects on the capacitation and acrosome status of frozen — thawed human sperm. While comparing the motility characteristics of reagent - treated cells and non - treated cells with CASA, however, we found that all these three reagents did modify the swimming behaviors of frozen — thawed human sperm. The results of our studies gave us some hints of mechanisms about the modulation of sperm fertilizing ability: After 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated cells are significantly greater than that of non — treated cells. This indicated that the intracellular cAMP level determines swimming velocity of sperm. Within the physiologically effective range, the higher the intracellular cAMP level, the larger the VCL. Because the swimming velocities of frozen — thawed cells did not be affected by the addition of FPP, we concluded that FPP, as a first messenger, may act on AC / cAMP signaling pathway indirectly. Despite of no effects on the velocities of the post — thawed sperm cells, FPP made the departure of cell tracks and paths significantly farther away from a straight line after 4 hours of incubation. Such a finding suggested that FPP is more closely than adenosine related to the alterations of swimming tracks associated with hyperactivation, which most of post- thawed sperm failed to express because of the damages resulting from freezing and thawing procedure. In addition, it is reasonable that the motility rates reflected the overall energy reserve of sperm population in our study. By activating adenylate cyclase, adenosine converted ATP to cAMP and thus contributed to the significant fall in the motility rate of post — thawed cells after 1 hour of incubation.. In contrast to pentoxifylline, which accelerated the movement of frozen — thawed cells without affecting motility rate, FPP caused a significant decrease in motility rate without speeding the sperm cells. There must be additional energy expenses other than those required for swimming in the FPP — treated cells. These additional energy expenses, which resulted in intracellular “ energy shift “, also accounted for the less active sports ( less BCF without greater velocities ) in the FPP —treated cells after 1 hour of incubation. After 4 hours of incubation, either of the adenosine — treated and FPP — treated groups had a smaller, but not significantly different motility rate from that of control group. This indicated that a substantial proportions of cryopreservation — damaged cells, because of energy exhaustion resulting from the addition of FPP or adenosine, did not survive as long as if not exposed to any reagent post — thawed. Sperm cell membrane plays an essential role in capacitation and acrosome reaction. Cryopreservation causes damage to the integrity and function of the sperm cell membrane and thus alters the permeabilities of ion and water, leading to sperm intracellular ion concentrations out of the physiologic ranges optimal for fertilization. It is well known that the intracellular Ca＋＋ concentration is closely correlated with the fertilizing ability of spermatozoa, and rearrangements of cytoskeletal elements modulated by intracellular Ca＋＋ / calmodulin — dependent protein kinases lead to modifications of cell movement. Because the previous literature revealed that FPP is a potential modulator of capacitation and hyperactivation, and our studies showed that FPP could modify swimming behaviors with additional energy expenses in frozen — thawed sperm cells with non - optimal intracellular ion concentrations, we propose that FPP potentiates fertilizing ability and prevents spontaneous acrosome loss, via regulating membrane — bound Na＋ - K＋ATPase and / or Ca＋＋ATPase, by keeping the sperm intracellular Ca＋＋ concentration within the “ capacitation “ range. In addition to cAMP, the intracellular Ca＋＋ is another second messenger in modulation of capacitation and acrosome reaction of mammalian spermatozoa. FPP failed to speed frozen — thawed cells because the intracellular “ energy shift “ resulted in no significant increase in the cAMP level. However, pentoxifylline also made the departure of cell tracks significantly farther away from a straight line as FPP did. Therefore, there may be “ cross — talk “ between Ca＋＋ / calmodulin and AC / cAMP signaling pathway. While acting on the specific receptor on the sperm cell membrane, FPP may make conformational changes of the cytoplasmic domain of its receptor which then activate G proteins. Some of these G proteins help modulate the intracellular Ca＋＋ level, activate the Ca＋＋ / calmodulin — dependent protein kinases, and thus lead to rearrangements of cytoskeletal elements associated with hyperactivation. Other G proteins modulate AC / cAMP and the consequent protein phosphorylation. Both Ca＋＋ / calmodulin and AC / cAMP signaling pathway interact with each other and thus modulate the expression of capacitation and hyperactivation. Although Green et al. reported that FPP induces capacitation in fresh human sperm within one hour of incubation, our study showed that the physiologic effects on the motion characteristics of frozen — thawed cells caused by FPP appeared beyond one hour of incubation. Such a delay resulted from a longer time it took for FPP to modulate the intracellular ion concentrations, which might be far away from the physiologic ranges, in the viable frozen — thawed sperm cells to the optimal levels for fertilization. Because cryopreservation disrupted sperm cell membrane and cytoskeleton, only a few “ healthier “ post — thawed sperm cells could express capacitation and hyperactivation. This is why none of the three reagents used in our studies made any difference in the fertilizing ability between reagent —treated and control groups. We suggest that FPP resets the intracellular ion concentrations of sperm cells at levels optimal for fertilization at ejaculation. Both the intracellular Ca＋＋ and cAMP act as second messengers in the modulation of sperm fertilizing ability. There may be branching and cross — talk between pathways involved in capacitation to regulate and coordinate a sperm cell’s response to specific signal molecules such as FPP and adenosine. It consumes energy to regulate the intracellular ion concentrations and to amplify the cell’s specific response to the incoming information, so is the physiologic significance of the phenomenon of “ capacitation “. Perspectives To verify the mechanism of FPP in modulating the fertilizing ability of mammalian sperm, we could try to use digitalis and vanadate to evaluate the possible effects of FPP on Na＋ - K＋ATPase and Ca＋＋ATPase on the sperm cell membrane. Digitalis is an inhibitor of Na＋ - K＋ATPase, which leads to an elevation of intracellular Ca＋＋ level. By stablizing the structure of Ca＋＋ATPase, vanadate slows down the passage of Ca＋＋. Comparisons of the effects of FPP, digitalis / vanadate, and FPP ＋ digitalis / vanadate on spermatozoa, we will clarify the possible mechanisms of FPP we proposed on the sperm intracellular ion concentrations. In addition, it was recently demonstrated that ceramide stimulates the Ca＋＋ATPase in a dose — dependent manner and sphingosine has conversely an inhibitory effect on Ca＋＋ATPase activity. Such sphingolipids may also be considered in assessing whether or not FPP has any effects on membrane — bound Ca＋＋ATPase of sperm. Calcium channel blockers such as nifedipine, verapamil, and diltiazem may also help us understand the role of calcium channel in capacitation and acrosome reaction. By using microarray, we may try to specify the proteins and their functions in the signal pathways responded to FPP and adenosine. Once proved to contribute to regulation of membrane — bound Na＋ - K＋ATPase and Ca＋＋ATPase, FPP could be widely applied in biomedical science.

碩士
國立屏東科技大學
動物科學與畜產系所
103
The objective of the present study was to investigate the effects of cryoprotectant composition, including various concentrations of glycerol, trehalose, and low density lipoprotein (LDL), and freezing rate on the quality of frozen-thawed boar semen. Parameters analyzed were sperm motility, viability, acrosome integrity, mitochondrial potential and DNA integrity. Results demonstrated that the semen quality of cyroprotectant containing 6% glycerol is significantly (P < 0.05) improved in comparison with those of croprotectant containing 3 and 4.5 % glycerol. Semen positioned 1 cm above the surface of liquid nitrogen (average freezing rate = -60℃/min) had significantly (P < 0.05) better quality then semen positioned 3 cm above the surface of liquid nitrogen (average freezing rate = -30℃/min) after thawing. Using the cyroprotectant containing 300 mM trehalose lead to significantly (P < 0.05) improved semen quality when compared to using cryoprotectants containing 250 mM trehalose, 350 mM trehalose, and 11% lactose. In addition, cyroprotectant containing 10% LDL was significantly (P < 0.05) better than 8% LDL, 12% LDL, and 20% egg yolk in terms of maintaining semen quality. Taken together, the combination of using the cyroprotectant containing 6% glycerol, 300 mM, and 10% LDL with the average freezing rate at -60℃/min (positioned 1 cm above the surface of liquid nitrogen) had the optimal semen quality. Using such semen for artificial insemination (AI) had similar (P > 0.05) conception rate (53.8 vs. 54.5%) in comparison with using fresh semen. However, the litter size (13.0 v.s. 9.2) and the number of piglets born alive per litter (12.0 v.s. 9.0) of the sows AI with frozen semen were significantly higher than those of the sows AI with fresh semen.

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.

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 (P=0.260), ovulation within 48 h of OvuGel® (P=0.411), and weight prior to breeding (P=0.681) did not influence conception rate, however expression of estrus was determined to significantly influence conception rate (P=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 (P=0.066).

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 (P<0.0001) and G:F (P<0.0001) compared to DS pigs. Pigs fed FIB resulted in reduced ADG (P=0.020) and reduced G:F (P=0.007). At slaughter LB pigs were 26.4 kg lighter than DS pigs (P<0.0001), and pigs that were fed FIB had lighter live weights (P=0.002) than pigs fed CON. LB pigs had 28.5±1.3 cm² smaller longissimus muscle area (P<0.0001), yielding 2.0 cm more 10^th rib back fat than DS pigs (P<0.0001). CON pigs had heavier HCW (P<0.0001) than FIB pigs, however FIB pigs had greater percent lean (P=0.015). LB pigs had significantly reduced percent lean than DS pigs (P<0.0001). LB pigs had loins with reduced drip loss (P=0.009) and cooked shear force values (P<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.

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.

Background It is hypothesised that an anatomical simulant model, that replicates the heterogeneous nature of human organs and tissues, will provide a more reliable and accurate method of evaluating the pathological features and incapacitation potential of ammunition in a weapons system than homogeneous bare ordnance gelatine alone. The use of frozen and thawed cadavers for simulant development was also examined. To develop a model, the most critical organs and tissues that sustain bullet wound trauma within the thorax and abdomen must be determined. Next a suitable method for establishing and matching the relevant biomechanical properties with candidate simulant materials must be developed, and an appropriate scoring system adopted. Method De-identified wound trauma data from 197 homicidal gunshot post mortem examinations in Israel were obtained between 2000-2001 and 2004-2008. The corresponding forensic ballistics data was only available for the cases between 2004 and 2008. The major organs involved, type of wounds, cause of death (COD), most common bullet paths, distances involved, firearm calibres and bullet types were established. Tensile strength tests were undertaken on selected tissue samples from an un- embalmed cadaver that had been frozen and thawed five times, which maximised the effects of repeated cycles. The universal test equipment Hounsfield H50KM machine was used to apply uniaxial tension until tissue failure occurred. The maximum tensile strength results in g/mm² were compared against corresponding data from the literature. Energy loss tests were conducted on fresh porcine organs/tissues using steel 4.5mm BB pellets fired from a Daisy® brand air rifle. Each organ/tissue was tested at room temperature and 37°C (body temperature). They were compared to Federal Bureau of Investigation (FBI) and North Atlantic Treaty Organisation (NATO) specification ordnance gelatine, as well as a candidate simulant material. A limited number of tests were also conducted at 4°C for further comparison purposes. Two chronographs measured BB pellet velocity before and after each test material was perforated and the difference was established in m/s. The resulting energy loss was established using the formula KE = ½ mv². FBI and NATO specified ordnance gelatine of 250 and 285 Bloom strengths were manufactured using tap water, reverse osmosis (RO) water and de-ionized water. They were allowed to cure for 21 hours, 100 hours and 3 weeks. The FBI calibration standard was used for all formulations as there is no separate standard for the NATO formulation in the literature. An Australian Defence Force (ADF) AUSTEYR model F88 ICW (individual combat weapon) in calibre 5.56x45mmNATO was used with standard issue ASF1 ball ammunition. Large FBI specification ordnance gelatine blocks were manufactured and thin gelatine/composite plates were used to simulate subcutaneous tissue and fat, as well as to provide a platform for the attachment of a skin simulant and to embed bone/rib composite within. A 250mm air gap and bubble wrap was used to simulate an expanded lung. The gelatine/composite plates were secured to a wooden cradle and the gelatine blocks were positioned behind it. The F88 ICW was fixed in a remote firing device 50m from the target and a chronograph 3m in front of the rifle measured bullet velocity. Test results were recorded using two high speed ‘Photron Fastcam’ digital cameras. Maximum three dimensional permanent cavity dimensions were obtained using a vernier gauge, and temporary cavity measurements were taken from high speed video images. Results The homicide study established that males represent 91% of gunshot victims. Of the 999 bullet wounds recorded, males were struck in the body an average of 5.2 occasions, with 2.2 of these bullets striking the thorax and/or abdomen. A contributing factor to the frequency of bullet strikes was the type of firearms involved, namely semi automatic pistols in the predominant calibre 9mm Luger, and assault rifles in calibre 5.56x45mm and calibre 7.62x39mmSoviet. Full metal jacket bullets were used in most instances and the majority of shootings (N=124) occurred at ranges estimated at 1m or greater. The most common bullet path was front to back in 66% of cases, followed by back to front in 27% of cases. Entry wounds occurred more often on the left side of the thorax, abdomen and back (N=253) compared to the right (N=172). The most common critical organs/tissues to sustain bullet trauma in descending order were; heart, lungs, liver, aorta, spleen, kidneys and vena cava. Ribs were struck by most bullets that entered the thorax. Multiple organ injury was listed in 146 of the 192 cases where a specific COD was determined by the pathologist. The following tensile strength results were achieved from the cadaver study: heart 3.56g/mm², kidney 10.27g/mm², oesophagus 22.08g/mm², skeletal muscle 29.46g/mm², ascending aorta 59.98g/mm², trachea 155.40g/mm², spleen 4.65g/mm², liver 10.83g/mm², pancreas 15.18g/mm², lung 29.94g/mm², pericardium 136.84g/mm², skin (abdomen) 355.26 g/mm² and skin (thorax) 407.88g/mm². These data were compared to published results obtained from non-frozen tissues from elderly persons, recognising that tensile strength values were only available for the following organs and tissues at the 95% degree of confidence: heart 9.2±0.95g/mm²; kidney 4±0.20g/mm², oesophagus 51±1.1g/mm², skeletal muscle 9±0.30g/mm², ascending aorta 68±2.4g/mm², trachea 150±6.5g/mm². It can be seen that some results from the test cadaver were higher and some lower than the published results, with trachea recording the only similar result. This indicates that the freezing and thawing process may change the tensile strength of tissues in unpredictable ways. Therefore, bio- mechanical research should avoid the use of frozen/thawed tissues and organs. The major agreement between the porcine energy loss tests were: FBI specification gelatine was similar (p>0.05) to heart and lung at room temperature and 37°C; spleen was similar to NATO specification gelatine at room temperature and 37°C; candidate Simulant ‘A’ was similar to hindquarter muscle at room temperature and 37°C and hindquarter muscle, kidney and spleen were similar to each other at room temperature and 37°C. Liver and kidney, and liver and fat were similar to each other at 4°C. The use of different water types had no effect upon ordnance gelatine calibration results. However, different temperatures, concentrations and curing times did have a significant effect. Neither of the two NATO 20% formulations met the same calibration standard as the FBI 10% formulation. The penetration depths achieved for the FBI formulations at both 3°C and 4°C were closest to the recommended calibration standard after 3 weeks curing time. A 20% concentration of 285 Bloom at 20°C met the same FBI calibration standard after 100 hours of curing and can be considered comparable. The anatomical model pilot tests demonstrated the benefit of using simulants that are more representative of the heterogeneous nature of human organs/tissues. It was found that by combining skin, bone and other simulant materials with ordnance gelatine, the behaviour of a military full metal jacket (FMJ) rifle bullet changes with regard to the earlier onset of temporary cavitation, reduced penetration depth and a higher degree of bullet yaw compared to simulations using only bare FBI specification ordnance gelatine. This occurs because more energy is consumed negotiating the various anatomical simulants, which means wounding is likely to occur much earlier, and organs that are deeper within the body may not be affected to the same degree. These factors will impact significantly upon injury severity in real tactical scenarios. Conclusion The experimental studies provide the framework for the development of a heterogeneous model for bullet trauma simulations of the thorax and abdomen. This model would be more representative of actual wound trauma than bare ordnance gelatine alone. This conclusion was arrived at by identifying the most critical organs/tissues for modelling purposes. Their energy loss values (J/m) were established and the method adopted allows for comparable simulants to be developed. Porcine energy loss tests showed that FBI specification gelatine is similar to heart and lung, but different to hind quarter muscle and most of the other ‘critical’ organs and tissues within the thorax and abdomen. NATO specification gelatine is a suitable simulant for spleen, and test Simulant ‘A’ is a suitable simulant for both hindquarter muscle and kidney. A separate simulant would be required for liver, fat and aorta. Frozen and thawed cadaveric tissue was shown to produce unpredictable tensile strength data and is therefore unsuitable for simulant development. The limitations of using FBI and NATO specification ordnance gelatine was highlighted when changes to bloom number, temperature and curing times altered calibration results. Therefore, temperature stable synthetic simulants such as Simulant ‘A’ are preferable. The anatomical model pilot tests clearly demonstrated that the addition of simulant materials directly affects wound severity simulations compared with bare ordnance gelatine alone. This in turn affects interpretation of real life situations. The AIS 2005/2008 and MAXISS scoring systems are deemed appropriate to grade the lethality potential of model simulations. Therefore, the original hypothesis has been validated.
Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2014.

碩士
逢甲大學
土木及水利工程研究所
81
This thesis is an investigation of no larger change in the coefficient of consoidiation of the frozen soil after thawing. The soil selected for this studies are saline soils which are popular in existence along the site of the coasts of Taiwan. The testing soils are sampled from the site of Taichung Fossil-fueled Power Plant ， Expansion Unit 2. Because this construction project is located near the seashore of Taichung Harbour ，the soils sampling contain in nature of 1％ salt concentration. In addition ，these salt-affected soils possess some amounts of the anode ions (sodium- and magnesium-ions). Due to occurrence of the factors， their influence to the coefficient of consolidation are also included for studies. According to their physical properties， we take four different soils for tests. The samples are : 1. No salt soil. 2. The saline soil of 1％ salt concentration. 3. The soil of 1％ sodium concentraction. 4. The soil of 1％ magnesium concentraction. And from the testing results，we conclude that: A. The coefficient of consolidation after thawing of these four sampling soils are almost close. B. The coefficient of thaw settlement Ａo varies with different soils ，it is examined that the sodium soil has relatively larger value than the regular soil. C. With temperature ranging from -15℃ to ambient 20℃,the void ratio will increase in all four sampling soils. But the regular soil will have the largest increase while the saline soil shows smallest increasing of void ratio. A full knowledge of the behaviors on the consolidation by freezing-thawing techniques will render a reasonable good solution to the engineers dealing with construction and design works.

Includes bibliographical references (leaves 232-257). 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.

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?