The health of somatic cell cloned cattle and their offspring

The cloning syndrome is a continuum with the consequences of abnormal reprogramming manifest throughout gestation, the neo-natal period, and into adulthood in the cloned generation, but it does not appear to be transmitted to subsequent offspring following sexual reproduction. Most in vivo studies on bovine somatic cell cloning have focused on development during pregnancy and the neo-natal period. In this paper, we report on the viability and health of cloned cattle in adulthood. From our studies at AgResearch, we find that between weaning and 4 years of age, the annual mortality rate in cattle cloned from somatic cells is at least 8%. Although the reasons for death are variable and some potentially preventable, the main mortality factor in this period is euthanasia due to musculoskeletal abnormalities. This includes animals with severely contracted flexor tendons and those displaying chronic lameness, particularly in milking cows. In contrast, no deaths beyond weaning have so far been encountered with the offspring of clones where the oldest animals are 3 years of age. In surviving cloned cattle, blood profiles and other indicators of general physiological function such as growth rate, reproduction, rearing of offspring, and milk production are all within the normal phenotypic ranges.     

Reproduction results and offspring performance after non-surgical embryo transfer in pigs

Increased interest in transfer of valuable genetic material around the world with minimal health risks has stimulated the development of non-surgical embryo transfer (nsET) technologies in pigs. Experimental evidence shows that nsET without sedation of the recipients is now feasible. The goal of this study, therefore, was to evaluate a method of nsET under commercial conditions. The experiment included 135 donor gilts and 45 multiparous recipient sows. Ovulation was induced in both donors and recipients, and nsET was performed using the Swinlet catheter. Donor gilts averaged 16.5 (7-45) corpora lutea, but this depended on age of the donor (P < 0.05). An average of 10.1 transferable blastocysts was recovered per donor, and the recovery rate was 84%. For 44 nsET, 14 recipients (31%) came into estrous before Day 23 after ovulation, 7 recipients (16%) came into estrous between Days 23 and 30, 3 recipients (6.8%) came into estrous between Days 39 and 48, 2 recipients (4.5%) had a late abortion. Finally, 18 of 44 recipients (41%) resulted in successful births, with an average liter size of 7.2 +/- 2.8. Birth weight of nsET piglets were 0.2 kg more than control piglets, but depended on litter size ((P < 0.05). The sex-ratio was not different from 50%. No anatomical abnormalities were observed in the offspring of nsET. Of the recipients that did not become pregnant from nsET, 91% became pregnant after insemination in the next estrous. Gilts born from nsET gave on average 12.4 +/- 3.0 total born piglets in their first pregnancy. In conclusion, the nsET procedure used in this study can be applied in practice without the need for special facilities, such as surgical and anesthesia equipment.     

Placentation in cloned cattle: structure and microvascular architecture

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.     

Ontogeny of cloned cattle to lactation

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.     

Evaluation of meat products from cloned cattle: biological and biochemical properties

Agricultural utilization of cloned livestock produced by nuclear transfer and their products for food will require public and governmental acceptance. A series of studies of properties of meat derived from cloned cattle was carried out to collect data for the safety assessment of cloned cattle products. Meat samples obtained from embryonic cloned, somatic cloned and non-cloned cattle were analyzed for chemical composition, as well as amino acids and fatty acids. Digestibility, allergenicity, and mutagenicity of meat were also examined. There were no significant differences in these properties among embryonic cloned, somatic cloned and non-cloned cattle. The analyses and tests revealed that there were no significant biological differences in meat from a non-cloned, an embryonic cloned, or a somatic cloned animal. A 14-week feeding trial in rats showed there were no abnormalities in body growth, general condition, locomotor activity, reflexes, sexual cycle, urinalysis, hematology, blood biochemistry, and histology. This study showed for the first time that the biological/biochemical properties of meat of cloned cattle are similar to those of non-cloned cattle.     

Health status of cloned cattle at different ages

The procedure of somatic cloning is associated with important losses during pregnancy and in the perinatal period, reducing the overall efficacy to less than 5% in most cases. A mean of 30% of the cloned calves die before reaching 6 months of age with a wide range of pathologies, including, for the most common, respiratory failure, abnormal kidney development, liver steatosis. Heart and liver weight in relation to body weight are also increased. Surviving animals, although mostly clinically normal, differ from controls obtained by artificial insemination (AI) within the first 1-2 months, to become undistinguishable from them thereafter. Hemoglobin concentrations, for instance, are lower, and leptin concentrations are elevated. In response to the lack of prospective studies addressing the health of adult clones, a long-term, 3-4-year study is currently being conducted to assess the health of mature bovine clones at INRA. Preliminary results over 1 year of study do not show any statistical difference between groups for hematological parameters.     

Production of cloned cattle from in vitro systems

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.     

Improving delivery and offspring viability of in vitro-produced and cloned sheep embryos

Recently developed, assisted reproductive technologies (e.g., in vitro embryo production and nuclear transfer) have encountered perinatal morbidity/mortality of the offspring produced, which are likely to hinder the application of these techniques. Consequently we have sought to develop a system of hormonal stimulation that will ensure the delivery of offspring more prepared for extrauterine life. Here we examine deliveries outcome in sheep carrying in vitro-produced and nuclear transfer (NT) embryos in comparison to artificially inseminated and naturally mated control ewes. All groups (excluding NT, which received one treatment) were subjected to one of two hormonal treatments for induction of delivery, whereas the third part of each group was left without any treatment. The first (commonly used for naturally mated ewes) dexamethasone treatment did not solve a majority of parturition disturbances, and actually the number of deliveries necessitating assistance was reduced (P < 0.05) by this treatment in the control group. On the other hand, combined estradiol plus betamethasone stimulation (E + B) solved a majority of complications regarding delivery performance such as lack of the preparation of the mammary gland, low myometrial contractility, insufficient cervical ripening, and impaired maternal behavior. Moreover, substantial reduction of neonatal mortality was observed following the combined treatment. In conclusion, the E + B induction of delivery overcame the majority of physiological and behavioral intrapartum failures of sheep foster mothers and increased the survival of offspring, and thus can be recommended as a safe method for inducing delivery in foster mothers carrying in vitro-generated embryos.     

Comparison of meat composition from offspring of cloned and conventionally produced boars

This study compares the meat composition of the offspring from boars produced by somatic cell nuclear transfer (n=4) to that of the offspring from conventionally produced boars (n=3). In total, 89 commercial gilts were artificially inseminated and 61 progressed to term and farrowed. All of the resulting piglets were housed and raised identically under standard commercial settings and slaughtered upon reaching market weight. Loin samples were taken from each slaughtered animal and shipped offsite for meat composition analysis. In total, loin samples from 404 animals (242 from offspring of clones and 162 from controls) were analyzed for 58 different parameters generating 14,036 and 9396 data points from offspring of clones and the controls, respectively. Values for controls were used to establish a range for each parameter. Ten percent was then added to the maximum and subtracted from the minimum of the control range, and all results within this range were considered clinically irrelevant. Of the 14,036 data points from the offspring of clones, only three points were found outside the clinically irrelevant range, two of which were within the range established by the USDA National Nutrient Database for Standard Reference, Release 18, 2005; website: (www.nal.usda.gov/fnic/foodcomp/search/). The only outlier was the presence of Eicosadienoic acid (C20:2) in one sample which is typically present in minute quantities in pork; no reference data were found regarding this fatty acid in the USDA National Nutrient Database. In conclusion, these data indicated that meat from the offspring of clones was not chemically different than meat from controls and therefore supported the case for the safety of meat from the offspring of clones.     

Chromosomal abnormalities: a potential quality issue for cloned cattle embryos

Nuclear transfer in cattle is associated with high levels of embryonic mortality and often with congenital malformation. Chromosomal abnormalities are a well-known cause of pregnancy failure and congenital malformation in humans, but their relative contribution to pregnancy failure and congenital malformation in cloned embryos and calves is largely unknown. This paper reviews existing literature on the chromosomal constitution of bovine embryos produced by fertilization in vivo and in vitro, parthenogenetic activation, and nuclear transfer. The published data suggest that chromosomally abnormal cells are common in embryos; however, the frequency reported varies with the method of embryo production. The most frequently observed deviation from the diploid karyotype was mixoploidy resulting from aberrant cell division causing polyploidy in a variable proportion of the embryo's cells.     

Assessing the quality of products from cloned cattle: an integrative approach

Scientific expertise was developed during a 3-year study to evaluate a large number of bovine female clones (n=37; from 4 to 36 months of age) and their products through a multidisciplinary approach and compare them to non-cloned breed, age and sex-matched contemporary control animals (n=38) maintained under the same conditions at the same experimental farm of INRA. In clone and control groups, most parameters measured for health and development of the animals as well as evaluation of milk and meat products were within the normal range for the breed. The strict comparison between cloned animals and controls allowed us to detect slight significant differences between the two groups. Cloned heifers reached puberty significantly later (+62 days) and at higher body weight (+56kg) than controls. There were slight differences in antigen-specific induced proliferation of lymphocytes after vaccination with ovalbumin before 10 months of age, but responses were normal responses in older animals. There were differences in the fatty acid (FA) composition of milk and muscle arising from two families of clones, suggesting a possible deviation in lipid metabolism as assessed by higher Delta-9 desaturase activity indices in both milk and muscle from clones compared to controls. Nutritional evaluation of milk and meat using the rat model did not reveal any difference between products derived from clones versus controls.     

Frequency and occurrence of late-gestation losses from cattle cloned embryos.

Nuclear transfer from somatic cells still has limited efficiency in terms of live calves born due to high fetal loss after transfer. In this study, we addressed the type of donor cells used for cloning in in vivo development. We used a combination of repeated ultrasonography and maternal pregnancy serum protein (PSP60) assays to monitor the evolution of pregnancy after somatic cloning in order to detect the occurrence of late-gestation losses and their frequency, compared with embryo cloning or in vitro fertilization (IVF). Incidence of loss between Day 90 of gestation and calving was 43.7% for adult somatic clones and 33.3% for fetal somatic clones, compared with 4.3% after embryo cloning and 0% in the control IVF group. Using PSP60 levels in maternal blood as a criterion for placental function, we observed that after somatic cloning, recipients that lost their pregnancy before Day 100 showed significantly higher PSP60 levels by Day 50 than those that maintained pregnancy (7.77 +/- 3.3 ng/ml vs. 2.45 +/- 0.27 ng/ml for normal pregnancies, P < 0.05). At later stages of gestation, between 4 mo and calving, mean PSP60 concentrations were significantly increased in pathologic pregnancy after somatic cloning compared with other groups (P < 0.05 by Day 150, P < 0.001 by Day 180, and P < 0.01 by Day 210). In those situations, and confirmed by ultrasonographic measurements, recipients developed severe hydroallantois together with larger placentome size. Our findings suggest that assessing placental development with PSP60 and ultrasonography will lead to better care of recipient animals in bovine somatic cloning.     

Genetic and epigenetic aspects of cloning and potential effects on offspring of cloned mammals

Although the biological mechanisms by which host cytoplasm and donor nuclei interact to produce a developmentally competent reconstructed embryo remain largely unknown, some advances have been made to our understanding of the genetic and epigenetic factors involved in the of reprogramming of the donor nucleus. Genetic alterations, which comprise changes to the genetic information in both the nuclear and cytoplasm compartments, are passed on to subsequent generations at fertilization and are a potential source of variation among cloned animals and their offspring. Apart from the major chromosomal anomalies found in developmentally arrested embryos and fetuses, less detrimental rearrangements and/or mutations are likely to go unnoticed in most donor cell karyotypes, suggesting that such problems could lead to inheritable anomalies among clones and their offspring. Mitochondrial DNA is also relevant to cloning because most animals inherit most or all of their mitochondria from the host oocyte. Epigenetic alterations to the DNA or to the histone packaging proteins are independent of gene sequences. Aberrant epigenetic events may lead to variable gene expression or mitosis and consequent effects on development and phenotype. Although much of the epigenetic marking is reset during embryogenesis and development, the impact of epimutations on progeny remains unexplored.     

Compositional analysis of dairy products derived from clones and cloned transgenic cattle

Cloning technology is an emerging biotechnological tool that could provide commercial opportunities for livestock agriculture. However, the process is very inefficient and the molecular events underlying the technology are poorly understood. The resulting uncertainties are causing concerns regarding the safety of food products derived from cloned livestock. There are similar concerns for livestock produced by biotechnologies which enable the purposeful introduction of genetic modifications. To increase the knowledge about food products from animals generated by these modern biotechnologies, we assessed compositional differences associated with milk and cheese derived from cloned and transgenic cows. Based on gross composition, fatty acid and amino acid profiles and mineral and vitamin contents, milk produced by clones and conventional cattle were essentially similar and consistent with reference values from dairy cows farmed in the same region under similar conditions. Whereas colostrum produced by transgenic cows with additional casein genes had similar IgG secretion levels and kinetics to control cows, milk from the transgenic cows had a distinct yellow appearance, in contrast to the white color of milk from control cows. Processing of milk into cheese resulted in differences in the gross composition and amino acid profiles; 'transgenic' cheese had lower fat and higher salt contents and small but characteristic differences in the amino acid profile compared to control cheese.     

Freshwater gomphonemoid diatom phylogeny: preliminary results

A cladistic analysis of eleven freshwater gomphonemoid diatom taxa yielded three equally most parsimonious cladograms. The three cladograms suggest Gomphoneis Cleve is non-monophyletic as originally and recently circumscribed. Gomphoneis elegans and G. transsilvanica (Pant.) Krammer are more closely related to Gomphopleura Reichelt ex Tempère than to other members of the genus. Gomphoneis geitleri Kociolek & Stoermer appears to represent a monotypic lineage. Among the taxa with differentiated apical pore fields Gomphonema kaznakowi Mereschkowsky appears to be primitively astigmate.The three cladograms vary with regard to systematic affinities among the Gomphoneis herculeana (Ehrenberg) Cleve group, Miocene gomphonemoid species from Idaho (USA), gomphonemoid taxa from East Africa and Gomphonema sensu stricto. Despite variability in number of puncta rows per stria and presence of an axial plate in the Miocene species from North America, inclusion of these taxa in the analysis provided better resolution of relationships than if they were excluded.     

Production of recombinant albumin by a herd of cloned transgenic cattle

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1–2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1–2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.     

Forearm elongation in gibbons: Hypothesis and preliminary results

The characteristic long forelimbs of gibbons are a consequence of elongation of all segments (hand, forearm, and arm) but especially the forearm. While the hand is limited in size by its prehensile role, both the arm and the forearm do not seem to be directly limited in a similar manner. Why is the elongation concentrated specifically in the forearm? One hypothesis, originally applied to cursorial ungulates, relates to the need to enhance angular velocity during the swing phase at a minimum cost by distributing the heaviest forelimb loads proximally near the shoulder joint. This appears to be a plausible explanation despite complicating factors associated with competing functions of the forelimb during the support phase of arm-swinging and the fact that the animal behaviorally adjusts inertial properties of its forelimb by flexing it while reaching for the support. In order to test this hypothesis, the forelimb was modeled as a series of uniform cylinders from which the radii of gyration (k) were calculated. After converting into relative values (%k), they were compared (1) interspecifically, among two hylobatids, an orangutan, common chimpanzee, and gorilla, and (2) hypothetically, among imaginary gibbons with brachial indices extending beyond the range found in hylobatids. The results of both tests were equivocal, suggesting that forelimb mass distribution may not be the dominant factor influencing elongation of the forearms in gibbons. Even though gibbons are unique in their great dependence upon arm-swinging, efficient generation of high angular velocities during the swing phase does not appear to be the overriding function beyond others related to support, propulsion, and prehension.     

Computer-aided hepatic tumour ablation: requirements and preliminary results

Surgical resection of hepatic tumours is not always possible, since it depends on different factors, among which their location inside the liver functional segments. Alternative techniques consist in local use of chemical or physical agents to destroy the tumour. Radio frequency and cryosurgical ablations are examples of such alternative techniques that may be performed percutaneously. This requires a precise localisation of the tumour placement during ablation. Computer-assisted surgery tools may be used in conjunction with these new ablation techniques to improve the therapeutic efficiency, whilst they benefit from minimal invasiveness. This paper introduces the principles of a system for computer-assisted hepatic tumour ablation and describes preliminary experiments focusing on data registration evaluation. To keep close to conventional protocols, we consider registration of pre-operative CT or MRI data to intra-operative echographic data.