Bovine in vitro produced embryos suffer from poor developmental competence and altered metabolism which hinders their cryotolerance. Overall, the goals of this thesis were to improve oocyte and embryo culture with the antioxidant polydatin and to optimize slow freezing procedures. This thesis was designed as three experiments, and in each experiment, oocytes were aspirated from abattoir ovaries, matured for 23h, fertilized with semen from 1 of 3 bulls, and cultured in synthetic oviductal (SOF) based medium (SCF1) in 38.5 °C in 5% O2, 5% CO2 and 90% N2. Stage 7 blastocysts were stained with Nile Red for lipid content or Mitotracker Red CMX-Rosamine for mitochondrial activity or Cell Rox Green for reactive oxygen species. Ten images per embryo were acquired using confocal microscopy at 5-µM step size and detected fluorescence by IMAGE PRO software. Embryos were also slow frozen in ethylene glycol and analyzed for post-thaw re-expansion after 24 and 48 hours. Experiment one was a 2x2x2 factorial design to test two culture media (SCF1 and a conventional SOF media), the use of a sucrose dehydration prior to slow freezing (2 min in 0 or 0.6 sucrose), and the length of equilibration in ethylene glycol prior to slow freezing (10 or 20 min). We determined that embryos cultured in SCF1 had increased blastocyst rate, mitochondrial activity, and cryotolerance and decreased lipid accumulation (pin vitro.

Cryopreservation of in vivo derived Jersey bovine embryos have resulted in a 10% lower pregnancy rate compared to other dairy breeds. Poor embryo survival after cryopreservation has been partially attributed to the high lipid content of Jersey embryos. In vitro-produced (IVP) bovine embryos have darker cytoplasm than their in vivo-derived counterparts because of higher lipid accumulation. High lipid accumulation is associated with impaired embryo quality. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. L-carnitine is required for transport of fatty acids from the intermembrane space of the mitochondria into the mitochondrial matrix to support the process of β-oxidation, and enhances ATP production. We hypothesized that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin + L-carnitine would reduce lipid content of IVP embryos and vitrification with embryo collapse would improve the cryosurvival of Jersey IVP embryos. The objectives of this experiment were (1) to analyze lipid content of in vivo and IVP Jersey and Holstein cattle embryos, (2) to evaluate the effect of forskolin + L-carnitine added to IVP culture media, and (3) evaluate Jersey IVP survival rates after three cryopreservation procedures. The factorial experimental design for objectives one and two used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forsk/L-C). In vivo produced embryos (n = 27 blastocysts) were collected from superstimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of 1.5mM L-carnitine during maturation and embryo culture with forskolin (10 µM) added at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data was analyzed by ANOVA, and means were compared using Tukey’s HSD. For the third objective, grade 1 Jersey IVP blastocysts (n=356) were divided into six treatments using a 2x3 factorial design comparing intact (IB) vs collapsed blastocoele (CB) and three cryopreservation methods: slow freezing (SF) vs vitrification using open pulled straws (OPS) or cryotop (CT). Slow freezing embryos were equilibrated in 0.7 M glycerol and 0.1 M galactose in holding media for 10 min, held for 10 min at -6°C, seeded after 5 min, decreased to -32 °C at 0.5 °C /min, held at -32°C for 5 min, and finally plunged into liquid nitrogen. Vitrified embryos were equilibrated in 1.5 M ethylene glycol (EG) for 5 min, exposed to 7 M EG + 0.6 M galactose for 30 s while loaded into OPS or placed onto CT, then immediately plunged into liquid nitrogen. SF embryos were thawed in air for 10 s and placed in a water bath at 37°C for 45 s. Vitrified embryos were warmed directly into holding medium at 37°C supplemented with 1.0 M, 0.5 M and 0.25 M galactose for 3 minutes each. Subsequently, embryos were cultured in BBH7 and re-expansion rates were assessed at 24 and 48h post warming and data was evaluated by GLIMX. For objective 1, Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin + L-carnitine lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. For experiment 2, re-expansion rates were higher for CT, than OPS, and SF (85 vs. 66 vs. 72% ± 0.4, respectively; p<0.05). Main effect means for re-expansion were higher for CB than IB (79 vs 68% ± 0.3; p<0.05). In conclusion, IVP embryos have higher lipid accumulation over Holstein in vivo embryos. Addition of forskolin and L-carnitine to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. The CT method and collapsing the blastocoele prior to cryopreservation resulted in higher blastocyst survival rate. Further studies including transfer of embryos to recipients are necessary to corroborate these results.

Equine spermadhesin HSP-7 is a 14 kDa protein isolated from stallion seminal plasma and present on the surface of spermatozoa. HSP-7 displays carbohydrate and zona-pellucida binding properties, but the physiological role in equine fertilization is not well defined. HSP-7 has 98% amino acid sequence homology with the well-studied boar spermadhesin, AWN. Currently, these two proteins are considered to have the same reproductive function. Immunofluorescence studies presented here show that the stallion and boar spermadhesins are localized to different segments on spermatozoa. The variation in molecular compartmentalization of spermadhesin molecules in different species suggests that these structurally related proteins could be involved in independent events of fertilization. While the variation in HSP-7 abundance was not statistically significant between fertile and subfertile stallions, capacitated spermatozoa displayed a marked increase in HSP-7 when compared to neat sperm (P < 0.05). These results indicate that rather than aiding in capacitation, HSP-7 is exposed with capacitation and may have a more significant role in the acrosome reaction and sperm-oocyte recognition than previously documented.

The influence of feeding juvenile female leopard tortoises (Geochelone pardalis, n=18) a commercially available, complete, extruded feed three (3) or seven days (7) per week on dry matter and digestible energy intake, apparent digestibility of dry matter, organic matter, gross energy and fiber fractions, animal body weight and measurements, digesta transit time, rate of passage, and indigestible fill was evaluated. Both feeding frequencies are commonly practiced with captive tortoises. When fed 7 compared to 3 days per week, dry matter and digestible energy intake was greater. Tortoises gained more g BW, but not when adjusted per kg initial BW. When fed 7 compared to 3 days per week, tortoises grew more in plastron width (PW) and carapace height (CH), but not midline straight carapace length (MSCL), and grew more in calculated shell volume (i.e., a calculated estimate of shell volume using MSCL, PW, and CH), with a higher calculated body condition index (BCI). Providing short fasts (i.e., feeding 3 compared to 7 days per week) may be useful in slowing tortoise growth when animals are provided food ad libitum. In general, ad libitum feeding, especially of a highly digestible extruded feed, is not recommended for captive juvenile G. pardalis, especially when offered food daily. With two data points (detected as outliers) removed due to low fecal output (and resulting unrealistically high apparent digestibility of all nutrients analyzed) of two animals when fed 3 days per week, apparent digestibility of cellulose in tortoises fed 7 (n=18) compared to 3 (n=16) days per week was lower, but no differences were detected in DM, OM, GE, or any other fiber fractions analyzed. Transit time (TT1) was shorter and indigestible fill was higher in tortoises (n=18) fed 7 compared to 3 days per week, regardless of percent Cr marker recovered. With four animals removed due to <50% Cr marker recovery, tortoises fed 7 compared to 3 days per week exhibited shorter mean retention time (RGIT), with no differences in digesta transit or indigestible fill. Longer digesta retention when food availability included short periods of fasting may have allowed tortoises to extract more energy from cellulose.

Since all chemical and electrical input into the cell / occurs via its plasma membrane, many pharmacological agents and hormones initiate their effects by interacting with receptor sites on the plasma membrane or by passing through the cell membrane and interacting with other membranes within the cell. Generally, the precise mechanisms by which these events occur remain relatively unclear. It was thought for many years that the cell membrane was a relatively static structure which allowed molecules to enter the cell at a rate dependent upon their degree of lipid solubility. However, more recently an increasing body of evidence suggests that cellular membranes are dynamic structures hosting many enzymatic activities and that many drugs or hormones can mediate intracellular effects via these membrane enzyme systems. ... Mammalian brain AChE from the caudate nucleus was chosen as the specific membrane protein to be investigated. Studies were initiated on the rationale that subcellular fractionation and the solubilization of AChE from membrane fractions under various experimental conditions would secure these general goals; (i) to obtain a stable aqueous solution of AChE of high specific activity and yield which would provide the basis of further purification and characterization studies, and (ii) to elucidate some of the basic physicochemical relationships of AChE to membrane components.

Very little is known of the development, life history, economic importance, of biological relationships of larval trematodes, especially in their molluscan and arthropod hosts on the Pacfic Coast. The comparatively few contributions to our knowledge have been from scattered and fragmentary observations. The purpose of this study was to expand our knowledge and to lay a foundation for further work on trematode life cycles in marine organisms. The area selected for special investigation was Nick's Cove, Tomales Bay, California.

A survey of the trematode parasites of marine fishes of Tomales and Bodega Bays, California, was instituted In the Spring of 1948. Many adult forms were obtained, but the search progressively narrowed Itself to a study of the larval trematodss of an area of Tomales Bay, known, as Nick's Cove. Here an interesting cystophorous cercaria was discovered in the littoral snail, Thais emarglnata.

During the summer of 1957 the writer became interested in water mites while collecting insects in an unnamed pond (see map in appendix) near Tomales, California. It was noted during that period that specimens of a giant water-bug, Lethocerus americanus Leidy (1847) had many larval mites attached to their ventral sides, and the lack of information concerning these animals and their life histories stimulated interest in investigating the problem detailed in this thesis. This work was begun in the summer of 1957 and has continued through the summer of 1959 and 1960. Due to the lack of information and lack of agreement by other investigators concerning the morphology of the adult Hydrachna miliaria Berlese (1888) [bilunata Marshall (1931)], the problem of this thesis is to redescribe it and to describe the stages of its life history. Stages of the life history of H. miliaria have not been described previously by other investigators.

This report evaluates vocal communication, nesting, territoriality, and other social behaviors of the California Scrub Jay.

Anemonala californica (Nutt.) Hook and Arn.(family Saururaceae), commonly known as Yerba del Hanza, is widely distributed in the United States and northern provinces of Mexico. The extract of the roots and rhizomes is used by the natives of Mexico in the treatment of rheumatic disorders, for asthma and also as a blood purifier.