D.C. Kraemer

A cat cloned by nuclear transplantation.

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Cell biology: A cat cloned by nuclear transplantation

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei.

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT‐PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1‐cell, 2‐cell, 6–8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK‐L, PFK‐M, and PFK‐P. PFK‐L and PFK‐P were not detected in bovine oocytes. PFK subunits were not detected in 6–8 cell embryos but were detected in blastocysts. Results from NT embryo RT‐PCR demonstrated that PFK was not detected in 8‐cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH‐A and LDH‐B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH‐C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH‐C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer. Mol. Reprod. Dev. 56:458–464, 2000.

Methods in bovine nuclear transfer

Abstract Numerous reports have been published which substantiate the feasibility of nuclear transfer in cattle, but procedural details are generally not presented. The objective of this paper and the demonstration which will be presented is to describe the equipment, methods and procedures which can be utilized for bovine nuclear transfer. The major items of equipment needed are micromanipulators, microscopes, plus electrofusion generator and chambers. Methods and procedures described include: preparation of microtools, cleaning of zona pellucidae, bisection of oocytes, transfer of blastomeres, fusion, and embryo culture. Each of these steps in the process is described in detail.

Intrauterine insemination of farmed fallow deer (Dama dama) with frozen-thawed semen via laparoscopy.

Estrus and ovulation of mature fallow does (n=155) on two North American farms were synchronized by intravaginal silastic devices containing 0.3 g progesterone (CIDR-type G) for 14 d. Each of 151 does received laparoscopic intrauterine inseminations of either 50x10(6) (n=125) or 25x10(6) (n=26) frozen-thawed spermatozoa, 65 to 68 h after CIDR device withdrawal. Four does received intrauterine inseminations per vaginam of 50x10(6) spermatozoa 68 to 69 hours after CIDR device withdrawal. Semen from crossbred Dama dama damaxDama dama mesopotamica sires was collected in New Zealand by electroejaculation. The overall pregnancy rate to artificial insemination, as assessed by rectal ultrasonography at Day 45, was 67.7%. The pregnancy rates for does receiving laparoscopic inseminations were 58.2% (Texas; 50x10(6) spermatozoa; n=79 does); 80.8% (Texas; 25x10(6) spermatozoa; n=26 does) and 76.1% (New York; 50x10(6) spermatozoa; n=46 does). Three of the four does receiving intrauterine inseminations per vaginam became pregnant to the frozen-thawed semen.

Embryo recovery from exercised mares

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 degrees C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F(2alpha) was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 degrees C to a mean of 39.9 degrees C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8+/-0.5 compared with 41.5+/-0.5mm diameter; P<0.05), and had an increased time from PGF(2alpha) administration to subsequent ovulation (8.47+/-0.337 compared with 9.27+/-0.294 d; P<0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P<0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P=0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.

Comparative studies of the Ethynyl Estrogens used in Oral Contraceptives. VII. Effects with and without Progestational Agents on Ultracentrifugally Fractionated Plasma Lipoproteins in Humans, Baboons, and Beagles *

Ethynyestradiol and mestranol, in doses ranging from 50 to 100μg/day, were given to women in 21-day cycles; baboons and beagle dogs received 1 and 4μg/kg/day in a similar regimen. After a number of such cycles, megestrol acetate, norethindrone acetate, or dl-norgestrel was given concomitantly. Protein, cholesterol, triglyceride, and phospholipid levels were determined in total plasma and in ultracentrifugally separated lipoprotein fractions. Over the dosage range studied, the effects of the two kinds of estrogen were indistinguishable. Except for human total plasma triglyceride, no dose-related differences were observed. The lowering of serum protein and the increase in cholesterol induced by estrogen were more pronounced in baboons and beagles than in human subjects. The cholesterol-depressing effect of progestational compounds observed in humans was very pronounced in baboons but absent in beagles. In all three species, estrogen increased the lipoprotein fraction cholesterol, except for human low-density lipoprotein cholesterol, which was decreased. Human plasma triglyceride and phospholipid increased on estrogen administration and were decreased by the progestins; in the two animal species, triglyceride is normally very low and the estrogen-induced changes were negligible; the phospholipid rose with estrogen but was unaffected by progestins. In sum, the two animal species show many similarities to, as well as important differences from, the human response of plasma lipids to various contraceptive steroids.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A

Summary.  Background: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. Objectives: To re‐establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Patients/methods: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. Results and conclusions: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non‐existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame‐shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Embryo collection and transfer in small ruminants

Abstract Recent studies of embryo transfer in small ruminants have yielded laparoscopic and nonsurgical procedures for both embryo collection and transfer. These relatively atraumatic methods for embryo collection produce results that are competitive with surgical methods for collecting uterine stage embryos. Embryos have been collected nonsurgically from sheep, goats, deer, suni antelope and the yellow-backed duiker. Laparoscopic embryo transfer is both rapid and effective when compared with surgical transfer. Although nonsurgical transfers have been achieved in goats, more data are required before this approach can be recommended for widespread application to small ruminants.