The low efficiency of the nuclear transfer (NT) procedure requires large number of oocytes to produce embryos and live offspring. A series of experiments were conducted to evaluate the ability of the bovine cytoplast to reprogram nuclei from horses and llamas. In a preliminary study, equine oocytes from small (<20mm diameter) follicles were either pretreated with roscovitine or placed in maturation (IVM only) prior to NT. Roscovitine pretreatment did not improve nuclear maturation rates (roscovitine pretreatment 57% vs. IVM only 66%) and no fusion was obtained from roscovitine-pretreated oocytes after NT. Another preliminary study was conducted with the objective to produce llama NT embryos and to compare their development in two in vitro culture conditions (G1.2® vs. CR1aa). No difference was found in the number of embryos cleaved after 2 d of culture. This resulted in the first scientific report of somatic cell NT, in vitro culture and transfer of NT embryos in the llama. In the next experiment, adult horse and llama fibroblasts were injected into enucleated cow oocytes. The results showed the cow cytoplasm is capable of partially reprogramming nuclei from other species and support mitotic divisions. However, this study also showed a consistent embryonic developmental arrest at the 8- to 16- cell stage when horse or llama donor cells were used as donor nuclei. When a more closely related species of donor cell (banteng) and recipient oocyte (domestic cattle) were used for NT, no embryonic developmental arrest was found. Embryos progressed to achieve high blastocyst rates (banteng male cell line 28% vs. banteng female cell line 15%). Two banteng interspecies NT pregnancies were established and subsequently lost from the banteng male cell line. In the final study, the effect of a mixed mitochondrial population (heteroplasmy) on early embryonic development was investigated. Ooplasmic transfer performed in combination with NT procedure indicated presence of foreign mitochondria clustered in a small portion of the cytoplasm in early stages of embryo development. When goat ooplasm was transferred into interspecies (cow oocyte-goat donor cell) NT embryos, fusion and cleave rates were reduced suggesting an increased level of heteroplasmy or nuclear-ooplasmic incompatibilities.

The interest in preserving endangered species has increased the amount of attention lent to the recovery of functional sperm from the epididymides of deceased males (Foote, 2000). Postmortem specimens have a finite time period before decomposition affects functionality. Determination of this window of opportunity to harvest and preserve epididymal sperm would be beneficial for further research in sperm preservation and assisted reproductive technologies. The objective of these studies was to determine 1) the window of opportunity to collect viable rat, caprine, equine and bovine epididymal sperm, 2) if epididymal sperm collected could be cryopreserved, 3) to test two common cryoprotectants for efficacy of sperm preservation, 4) to determine if bovine samples could be used to produce in vitro fertilization (IVF) embryos and 5) to establish if sperm subjected to a series of freeze-thaw cycles can maintain motility. Epididymal sperm collected from rat, caprine, equine and bovine males all maintained some level of acrosomal membrane integrity up to 96 hours postmortem. The bovine, caprine and equine sperm survived cryopreservation and exhibited greater preservation with milk-based extenders. In vitro fertilization with cryopreserved bovine epididymal sperm was not efficient but development of embryos proved limited usefulness. Finally, subjecting the bovine sperm to repeated freeze-thaw cycles proved extremely damaging and should be practiced only when absolutely necessary. Rat sperm exhibited a difference from 24 to 48 hour with a percent progressive motility (PPM) of 46 to 28%. Caprine sperm PPM and percent intact acrosomes (PIA) declined after 24 hours from 68 and 66% to 56 and 55% at 48 hours, respectively. Equine sperm exhibited a drop in PPM and PIA at 48 hour of 42 and 71% to 34 and 68% at 72 hours, respectively. Bovine sperm PPM dropped initially from 65 to 49% at 24 to 48 hours and again from 46 to 30% at 72 to 96 hours. The difference in PIA only appears between 24 and 48 hours of 77 to 65%. As stated previously, the epididymal sperm collected from the rat, caprine, equine and bovine males maintained acceptable levels of PPM and PIA up to 96 hours postmortem.

This research was conducted to determine the non-phosphorus effects of phytase in diets for swine and poultry. An experiment was conducted to determine the effect of phytase addition on energy availability and protein and fat deposition in growing pigs. Results from this experiment indicated that phytase had small positive effects on energy availability, protein deposition, and fat deposition. In this study, 23 of 29 response variables were at least numerically increased with phytase addition. Two experiments were conducted to determine the accuracy of the energy and amino acid matrix values for Natuphos 1200 in broilers from 0 to 21 or 0 to 42 d posthatching. Results from these experiments indicated that the nutrient matrix values for phytase are accurate, and that they can be used in diet formulations for broilers. Experiments also were conducted to determine the effect of phytase addition to diets with or without the trace mineral premix in nursery, growing, and finishing pigs and in commercial broilers. Results indicated that phytase can replace the trace mineral premix in swine diets. Phytase addition had no negative effect on growth performance or pork quality, and it had minimal effects on carcass traits. In broilers, removing the trace mineral premix had no effect on growth performance but decreased bone breaking strength, and adding phytase did not reverse this response. This research indicates that phytase addition has little effect on carcass traits or meat quality in swine and poultry. When formulating swine diets with phytase, the trace mineral premix can be removed with no negative effects on growth performance or pork quality. However, more research is needed to determine the effect of phytase addition in diets without the trace mineral premix in broilers, because the addition of phytase did not overcome the decrease in bone breaking strength.

The objective of Experiment 1 was to determine the effect of bovine follicular fluid (bFF) on nuclear maturation. Treatment A (Control) oocytes were stained with Hoechst-33342 immediately after aspiration from follicles, whereas, oocytes in Treatment B were held in bFF for 12 hours at 38¢ªC and then stained to determine nuclear status. No significant difference was detected between treatment groups. Results indicate that bFF inhibits resumption of meiosis. The objective of Experiment 2 was to determine the effect of bFF on embryonic development. Oocytes in Treatment A (Control) were placed into in vitro maturation (IVM) for 22 hours followed by in vitro fertilization (IVF). Oocytes in Treatment B were held in bFF for 12 hours at 22¢ªC, followed by IVM and then subjected to IVF. Significantly more (P<0.0001) oocytes cleaved, developed into blastocysts and hatched in Treatment A compared with Treatment B. Results indicate that a 12-hour holding period in bFF does not promote normal embryonic development. The objective of Experiment 3 was to determine the effect of decreased time and concentration of bFF on embryonic development. Treatment A (Control) oocytes were placed into IVM followed by IVF. Oocytes in Treatment B were held in bFF, oocytes in Treatment C were held in Lactated Ringer¡¯s Solution (LRS) and oocytes in Treatment D were held in a combination of bFF and LRS for 6 hours at 22¢ªC, followed by IVM then by IVF. No significant difference was detected between Treatments A and B when analyzing cleavage, blastocyst formation and hatching rates. However, significantly fewer (P<0.0001) embryos reached these stages of development in Treatments C and D. Nevertheless, there were significantly more embryos that developed to the blastocyst stage in Treatment D compared with Treatment C. Decreasing the amount of time that oocytes were held in bFF proved to be beneficial in supporting in vitro embryo production (IVP). These findings could be advantageous when attempting to rescue valuable gametes from deceased females.

Bakso is a finely comminuted boiled Indonesian meat product that is traditionally made of starch, salt and emulsified prerigor or early postmortem meat and often sold from street vendors. Recently processors have begun to commercially manufacture bakso. This research was conducted to investigate the substitution of early postmortem meat with postrigor meat to allow more efficient manufacturing and raw material procurement. The first experiment was to determine the properties of bakso with three tapioca starch concentrations added to early or late postmortem beef. No differences (p<0.05) were observed in bakso properties of composition and texture, though bakso made of post-rigor meat had slightly less elasticity, strength and shear. These disadvantages were compensated by incorporating 15% starch concentration, indicating that the replacement of early postmortem meat with postrigor meat was applicable in industrial mass production of bakso. The second phase of experiments was to investigate the properties of bakso with different frozen storage times of raw postrigor meat and after different times of frozen storage for bakso made from postrigor and early postmortem meat. Postrigor meat substitution for early postmortem meat in bakso with 15% tapioca starch resulted in minimum composition and textural differences (p>0.05). Meat stored frozen for 2 and 4 months was still suitable as raw material for bakso production. The decreased oxidative stability in bakso made from postrigor meat after frozen storage of 2 and 4 months should be addressed with antioxidant ingredients or procedures to minimize potential off-flavors of the precooked bakso products stored frozen.

The experiments described herein were designed to answer questions that arose from initially attempting to determine whether treatment with dexamethasone increased concentrations of leptin in geldings. Dexamethasone treatment did in fact increase leptin concentrations in mares, geldings, and stallions. Additional experiments were designed to determine leptin's interaction, not only with the adrenal axis, but with the thyroid axis, the growth hormone/insulin-like growth factor-1 axis, as well as glucose and insulin metabolism in geldings, mares, and stallions. During the course of these experiments, differences in leptin concentrations in the horse were attributed to degree of body condition, gender, and feeding time. Additionally, it was found that horses with high body condition fell into two distinct groups based solely on circulating concentrations of leptin (high vs low). The obese, hyperleptinemic horses were found to be hyperglycemic and hyperinsulinemic and had elevated concentrations of triiodothyronine and decreased concentrations of growth hormone, a hormonal profile similar to that of a type II diabetic human. Indeed, this obesity-related hyperleptinemia was associated with a degree of insulin insensitivity evidenced by the increased insulin response to glucose in these horses. Thus, further experiments were conducted to determine the degree to which these horses were insensitive to insulin, as well as whether diet supplementation or feed restriction might alleviate this insulin insensitivity. Chromium propionate supplementation did not decrease plasma insulin or leptin; however, restricted nutrient intake (6 h of grazing per day) was successful in decreasing concentrations of leptin. It was concluded that leptin in the horse is affected by adrenal and thyroid hormones as well as by glucose/insulin metabolism, and that a syndrome of obesity-related hyperleptinemia, hyperglycemia, and hyperinsulinemia exists in the horse that is similar to type II diabetes in humans.

The purpose of this research was to compare dietary inorganic and organic Se sources in poultry. An experiment was conducted to assess daily egg production and Se deposition in eggs of hens fed diets supplemented with inorganic or organic Se. The results from this experiment indicated that hen production was not affected by Se source, and organic Se increased egg Se concentrations more than inorganic Se. Two experiments then were conducted with broilers to compare dietary organic and inorganic Se on growth performance, carcass traits, breast and plasma Se concentrations, and plasma glutathione peroxidase activity. The results from these experiments indicate that organic Se increases tissue Se concentration more than inorganic Se, but plasma glutathione peroxidase activity, growth performance, and carcass traits were not affected by source. Finally, three experiments were conducted to develop a Se-deficient diet, and then to compare plasma glutathione peroxidase and plasma and tissue Se concentrations in broilers fed a Se-deficient diet after they had been fed diets supplemented with either inorganic or organic Se. The results from these experiments indicate that organic Se increases tissue Se concentration compared with inorganic Se, but plasma glutathione peroxidase activity and growth performance were not affected by source. However, when broilers were placed on the Se deficient diet, organic Se maintained plasma glutathione peroxidase activity longer and at a higher level than inorganic Se. Overall, the results from these experiments indicate that dietary inorganic and organic Se result in similar growth performance and plasma glutathione peroxidase activity in broilers and layers, but organic Se increases tissue and egg Se concentrations more than inorganic Se.

Four experiments were conducted to evaluate the effects of diets with reduced Ca or nonphytate P (nPP) levels with supplemental virginiamycin (Vm) on growth performance and bone response variables in chicks. All diets were corn-soybean meal based and all treatments were replicated six or eight times with five or six chicks each. Reducing dietary nPP from 0.45 to 0.15, 0.25, or 0.35% decreased most growth and bone response variables. The addition of Vm to diets containing 0.35% nPP and above increased both growth and bone response variables. The addition of Vm to diets containing 0.25% nPP and below had no affect or decreased growth and bone response variables. Reducing dietary Ca levels from 1.0 to 0.80 or 0.70% decreased bone response variables, and the addition of Vm increased these response variables at 0.70% Ca but not at 0.80% Ca. Another experiment was conducted to determine the effect of supplemental Zn, Mn, and Cu in the inorganic or organic forms (metal-amino acid complexes) on egg production and egg quality during the post-molt production period. Nine hundred and sixty post-molt layers, were randomly assigned to five treatments with 12 replications of 16 layers each. The study lasted 32 wk and was divided into eight periods of 28 d each. The treatments consisted of: 1) Control (C) supplemented with inorganic trace minerals; 2) C + organic Zn (40 ppm), Mn (40 ppm), and Cu (7 ppm); 3) C + organic Zn (40 ppm); 4) C + inorganic Zn, Mn, and Cu to equal Diet 2; 5) C + inorganic Zn equal to Diet 3. The addition of trace minerals had variable effects on the egg production and quality during different periods. However, in the overall data, none of the forms of trace mineral supplementation affected any response criteria.

There has been great controversy involving progesterone (P₄) levels during early pregnancy in cattle. The objectives of these experiments were to determine the effect of an early low dose administration of P₄ or altrenogest (ALT) on pregnancy rates in repeat breeder (RPB) females, if an increase in pregnancy rates could result from a direct effect of P₄ on the embryo and if ALT could support pregnancy in the absence of a functional CL. Firstly, ALT was evaluated for use as a progestin in cattle by synchronizing estrus in beef heifers. There were no differences in the number of females displaying behavioral estrus or in pregnancy rates when synchronized with ALT or MGA. A second experiment was designed to determine the effect of P₄ or ALT supplementation during days 3 to 5 on pregnancy rate in RPB cattle. It was determined that 15 mg of P₄ or ALT during days 3 to 5 increased pregnancy rates compared with nontreated breeding periods. A third experiment was designed to determine if P₄ exerted a direct effect on the embryo. In vitro produced (IVP) embryos were cultured in the absence of a co-culture system. At day 3, post-insemination, embryos were cultured in the presence of P₄ and evaluated on days 6 to 9. On day 7 post-insemination, there were significantly more grade 1 blastocysts from the P₄ group compared with other treatment groups. Also, embryo developmental rates were increased when cultured in the presence of P₄ and more of these embryos developed to the hatched blastocyst stage compared with other treatment groups. After a direct effect of P₄ on developing IVP bovine embryos existed, it was determined that these embryos did not possess P₄ receptors. Finally, it was demonstrated that ALT could support pregnancy in the absence of a functional CL. These experiments demonstrated that ALT could serve as a progestin in cattle and when administered in low doses during early pregnancy could improve pregnancy rates in RPB cows. These results are likely due to a direct effect of P₄ on the embryo; however, this mechanism is by means other than binding the PR.

Calving rates are significantly reduced following in vitro production of embryos. Thus, if a technique could be developed that would increase calving rates by as little as one viable offspring, significant research advances could be made. Therefore, in a series of experiments, the efficiency and quality of culturing IVP bovine embryos in the amnion of a domestic chicken egg was tested. In Experiment I, by culturing IVP bovine embryos in the chick amnion (day 4 to 7 of incubation) it was discovered that there was no significant difference in blastocyst rates compared with controls. In Experiment II, it was shown that nuclear transfer bovine embryos cultured in the chick amnion reached the blastocyst stage at rates equal to controls and were capable of producing pregnancies following transplantation into recipient females. In a subsequent experiment, a method of naturally improving the chick embryo co-culture (CEC) system was explored by treating the developing chick embryo with prostaglandin E₂ (PGE₂) or prostaglandin F_2α (PGF_2α). It was determined that treatment with PGE₂ increased angiogenesis within the developing chicken egg, while treatment with PGF_2α decreased angiogenesis. When bovine embryos were cultured in PGE₂-treated chicks, developmental rates were not increased. In Experiment IV, chick amniotic fluid (CAF) was evaluated as a media supplement to the control culture system. Although replacing fetal bovine serum (FBS) with CAF resulted in significantly fewer blastocysts on day 7 of culture, there were no significant differences in the number of grade 1 embryos between the two treatments. This finding was important because it demonstrated an ability to culture IVP bovine embryos in the absence of FBS, a medium component that has been implicated in numerous fetal and calf abnormalities. Another experiment was designed to develop a method of culturing cells derived from the chick embryo and surrounding amniotic membrane for later use as a co-culture system for bovine IVP embryos. In the final experiment, using a novel method to detect apoptotic cells, it was determined that CEC did not alter the number of apoptotic cells in the embryo when compared with in vivo-derived or in vitro-cultured bovine embryos.