Francis C. Gwazdauskas

Embryo Density and Medium Volume Effects on Early Murine Embryo Development

BackgroundOne-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro.MethodsEmbryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-µl drops of CZB under silicon oil at 37.5°C in a humidified atmosphere of 5% CO2and 95% air.ResultsDevelopment score for embryos cultured in 10 µl was higher than that of embryos cultured in 20 or 40 µl. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-µl drop. The percentage of live embryos in 20 or 40 µl was lower than that of embryos cultured in 10 µl. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups.ConclusionsOur results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1∶1 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

Effect of low dose of FSH given at the beginning of the estrous cycle and subsequent superovulatory response in Holstein cows

A total of 47 superovulations were conducted on forty non-lactating cows to evaluate two different schemes using follicle stimulating hormone (FSH) for superovulating cattle. Cows randomly assigned to treatment A (26 collections) were superovulated beginning on days 9 to 13 of the estrous cycle by giving FSH at decreasing doses of 6, 6, 5, 5, 3, 3, and 2, 2 mg for 4 consecutive days at 12-h intervals while those in treatment B (21 collections) also received 2.5 mg of FSH on days 3 and 4 of the estrous cycle. Animals in both treatments were each given 12.5 mg of prostaglandin F2alpha (PGF2alpha) at 60 and 72 h after the initiation of superovulatory treatment. Cows were artificially inseminated at 0, 12, and 24 h after the onset of estrus. Embryos were recovered nonsurgically on d 6 and morphologically evaluated. Ovaries of the cows were palpated at the end of flushings to assess the number of corpora lutea (CL). The mean interval from PGF2alpha to the onset of estrus was not different (P>0.05) for treatments A (56.6 h) and B (50.0 h). Also, mean duration of standing estrus was not different for either treatment (13.4 h vs 12.8 h). The mean number of CL palpated (7.3 vs 12.9) and ova recovered (5.5 vs 14.2) were significantly greater (P<0.05) for treatment B. The mean number of excellent and good embryos recovered was lower for treatment A animals, but not significant (P>0.05). Therefore, low doses of FSH given at the beginning of the cycle increased ovulation rate and embryo recovery in non-lactating cows.

Dietary and Seasonal Influences on Nutritional Indices of Adult Male White-Tailed Deer

Nutritional and seasonal influences on physiological indices of nutritional status were determined in a 1-year experiment with 10 male white-tailed deer (Odocoileus virginianus). The deer were placed on ad libitum or 0.75 ad libitum diets and sampled every 28 days. Voluntary feed intake decreased in October. Body weights peaked in October, reached their lowest level in April, and were greater for ad libitumthan restricted-fed deer. Hemoglobin concentration, packed cell volume, and mean corpuscular hemoglobin content (MCHC) varied monthly; only MCHC was significantly lower in restrictedthan in ad libitum-fed deer. Interactions between study months and diets were observed for blood urea nitrogen and urinary urea nitrogen/creatinine ratios. Significant seasonal variation was observed for serum cholesterol, regardless of level of feeding. Ketone-body concentrations in serum were greater for ad libitumthan restricted-fed deer during the last 4 months of the study. J. WILDL. MANAGE. 45(4):926-936 The use of physiological indices of nutritional status in wild cervids has received much attention in wildlife science. However, before these nutritional indices can be reliably and accurately applied in the field, controlled experiments must be conducted to identify and minimize environmental influences that may lead to alterations in physiological characteristics. Uncontrolled observations may lead an investigator to erroneous conclusions. Kirkpatrick et al. (1975) and Seal et al. (1978a) examined nutritional indices of white-tailed deer fawns under controlled conditions. However, Kirkpatrick et al. (1975) examined only blood urea nitrogen (BUN), and Seal et al. (1978a) conducted their experiment for only 10 weeks. Long-term experiments, similar to that of Bahnak et al. (1979), permit the determination of seasonal variations in physiological indices and thus provide additional valuable information. The objective of our experiment was to identify variations in nutritional indices of adult m le white-tailed deer in response to season and a 25% dietary restriction in a 1-year study. This study was supported in part by a grant from the Pratt Animal Nutrition Foundation at Virginia Polytechnic Institute and State University. Technical assistance was provided by D. Gibson, T. Jones, W. Morehead, J. Dickinson, and H. Warren. U. S. Seal provided assistance in developing the nonesterified fatty acid procedure.

Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos

The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3M sucrose solution at 37 degrees C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P<0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5+/-4.4% in VS-1 and 57.9+/-4.5% in VS-2; mean+/-S.E.M.) and 2-cell embryos (63.1+/-4.4% in VS-1 and 59.2+/-4.3% in VS-2) developed into blastocysts, development of control embryos (70.2+/-5.0% of zygotes and 75.5+/-4.4% of 2-cell embryos) into blastocysts was higher (P<0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.

Effect of gossypol on bovine embryo development during the preimplantation period.

The purpose of this study was to evaluate the effect of varying doses of gossypol acetic acid on early bovine embryo development in vitro. One hundred and forty-eight excellent and good quality bovine morulae were randomly cultured in 0, 1.0, 5.0, 10.0, 30.0 mug gossypol acetic acid (GAA) in normal steer serum and Hams F-10 media. Bovine embryo development was assessed at 12-h intervals for 96 h. Sixty-seven percent of embryos developed in 0 mug GAA to the hatched blastocyst stage, while 43, 19, 4 and 0% had comparable development in 1.0, 5.0, 10.0 and 30.0 mug GAA, respectively. Embryos in 5.0 mug GAA had a delayed development to the blastocyst stage compared to embryos in 1.0 mug GAA. Development time to expanded blastocyst stage was longer for 10.0 mug GAA embryos than 0, and 1.0 GAA-treated embryos. No embryo cultured in 30.0 mug GAA advanced past the morula stage. Final developmental scores were highest for embryos in 0 mug GAA (4.06) and lowest for embryos cultured in 10.0 and 30.0 mug GAA (0.44 and -0.02, respectively). Embryos cultured in higher doses of GAA degenerated sooner than embryos cultured in 0 mug GAA. These data show a dose-dependent detrimental action of GAA on early bovine embryo development and suggest a direct action on the embryo itself.

The Porcine Mammary Gland as a Bioreactor for Complex Proteinsa

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Peripheral plasma testosterone concentration and sexual behavior in young prenatally stressed boars

Abstract The purpose of this study was to examine the effects of stress induced physiological changes in the gestating sow on postnatal sexual and endocrine development of male offspring. Ten boars, ranging from 160 to 185 days of age, were randomly chosen from sows which had been maintained under either stress or control conditions during mid-gestation. Blood samples were collected weekly from each boar (minimum of four weeks) at 30 min intervals over a common six-hour period via an indwelling anterior vena cava cannula. Plasma testosterone concentrations were determined by radioimmunoassay. In order to ascertain degree of sexual behavior, boars were exposed weekly to gilts in estrus and a subjective score assigned. No differences (P>.10) were found between prenatally stressed and control boars in overall mean testosterone concentration or libido score. A significant (P

Anthropometric and Leptin Changes in Women Following Different Dietary Approaches to Weight Loss

Leptin may favorably respond to fat mass (FM) losses induced by a low‐carbohydrate (LC) diet, although this is unclear. We examined serum leptin concentrations in women in midlife undergoing different dietary approaches to body weight (BW) loss. Women followed either a LC, high‐protein (LCHP; n = 13) or high‐carbohydrate, low‐fat (HCLF; n = 12) diet for 12 weeks. Changes in anthropometric and soft‐tissue mass measurements and leptin concentrations were assessed. Women in both diet groups had reductions in BW, BMI, fat‐free soft‐tissue mass, FM, body fat percentage, and central abdominal fat (CAF) (P < 0.001 for all variables) over the 12‐week intervention. These changes were not significantly different between diet groups. Serum leptin concentrations decreased by 41.8% (P < 0.001) in the LCHP group and by 44.3% (P < 0.001) in the HCLF group from baseline to week 12, with no significant difference between groups. The association of CAF (r = 0.73) and FM (r = 0.83) change with leptin change was strong in the HCLF group. Leptin change did not relate to change in any variable in the LCHP group. Both LCHP and HCLF diets favorably lower FM, CAF, and leptin in women, suggesting that beneficial changes in leptin can be similarly achieved through different dietary approaches to BW loss.

In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39°C

Embryos from two strains of mice were used to assess the effect of incubation temperature on pronuclear and twocell development to the morula/blastocyst (M/B) stage. Embryos from B6D2F2 and B6SJLF1 strains were cultured in medium M16 at either 37 or 39°C until 120 hr post human chorionic gonadotropin (hCG) or 0, 24, or 48 hr at 37°C and the remaining time at 39°C. Overall M/B development for pronuclear embryos was 0.6, 0, 32.3, and 52.4% for 0—96, 24—72, 48—48, and 96—0 hr at 37 and 39°C, respectively. Only 0—96 and 24—72 hr at 37 and 39°C were not different (P >0.10). Overall M/B development for two-cell embryos was 48.1, 78.1, and 98.0% for 0—72, 24—48, and 72—0 hr at 37 and 39°C, respectively. Percentage development at each time was different (P <.01) for each category. Additionally, the number of nuclei for morulae and blastocysts tended to be higher for embryos initiating culture at the two-cell stage compared to pronuclear embryos. The first cell cycle was most dramatically affected by a 2°C increase in incubator temperature. More advanced embryos can tolerate slight increases in incubator temperature more readily than pronuclear embryos.