Xavier Vignon

Frequency and Occurrence of Late-Gestation Losses from Cattle Cloned Embryos

Abstract 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.

Lymphoid hypoplasia and somatic cloning

BACKGROUND Adult somatic cloning by nuclear transfer is associated with high rate of perinatal mortality but there is still no evidence that nuclear transfer itself is responsible for these failures. We report on a longlasting defect linked to somatic cloning. METHODS Skin cells grown from an ear biopsy specimen from a 15-day-old calf were used as a source of nuclei. The donor animal was a clone of three females obtained from embryonic cells. Clinical examination, haematological, and biochemical profiles, and echocardiography of the somatic clone were done from birth to death. FINDINGS After 6 weeks of normal development, the somatic cloned calf had a sudden and rapid fall in lymphocyte count and a decrease in haemoglobin. The calf died on day 51 from severe anaemia. Necropsy revealed no abnormality except thymic atrophy and lymphoid hypoplasia. INTERPRETATION Somatic cloning may be the cause of long-lasting deleterious effects. Our observation should be taken into account in debates on reproductive cloning in human beings.

Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells

Muscle and skin biopsies taken from bovine fetuses and young calves have been used as a source of donor nuclei for cloning experiments. After culture, cells were individually fused to enucleated matured oocytes and the resulting blastocysts obtained after 7 d of culture (3-8% depending on the cell type) were transferred to foster recipient heifers. Two calves, a female and a male, both originating from muscle cells were born, and four additional pregnancies have surpassed mild-term gestation. The pregnancies include one fetus established from a transgenic nucleus from a fetal skin cell, and another one resulting from a skin biopsy performed on a female calf. Our data demonstrate that nuclei from cultured bovine somatic cells obtained from differentiated tissues can be made multipotent.

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

BackgroundRecent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far.MethodsWe used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1), and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1), and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA).ResultsUnlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage.ConclusionOur results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

Effects of high-intensity high-frequency ultrasound on ageing rate, ultrastructure and some physico-chemical properties of beef

High-intensity and high-frequency ultrasound was tested for its ability to accelerate meat ageing and increase beef tenderness. Samples (≈50g) of semimembranosus muscles from 8 cull cows were assigned to ultrasonic treatment (2.6MHz; 10W/cm(2); 2 ×15s) either pre-rigor (day 0, pH 6.2) or post-rigor (day 1, pH 5.4). When applied pre-rigor, ultrasound induced a slight delay in rigor mortis onset, a stretching (12-15%) of the sarcomeres (p<0.05), an ultrastructural alteration in the Z-line region and an immediate increase (around 30%) in the release of calcium in the cytosol (p<0.05). However, no conclusive effect on meat ageing rate was observed. Post-rigor ultrasonic treatment did not induce any structural modification but slightly improved the ageing index after 6 days (p<0.05). However, no improvement in the final (day 14) ageing index was observed compared to the controls. As ultrasound had also no effect on the thermal stability of collagen, at both postmortem times, no improvement in meat tenderness can be expected under the conditions used.

Tenderization of beef by lactic acid injected at different times post mortem

The potential to tenderize beef muscles by the injection of lactic acid (0.5 M, 10% w/w) was studied using the pectoralis profundus muscle from cull cows. The injection was performed either 1 h (pre rigor) or 24 h (post rigor) post mortem, and the meat was stored for 2 or 14 days post mortem. Both treatments caused a rapid pH drop to around 5.0 within 4 h of injection. Other effects were: (1) an accelerated release of lysosomal enzymes into the cytosol; (2) a greater degradation of myosin heavy chains; (3) ultrastructural alterations of the myofibrils which included a general weakening or rupture in the M-lines and, to a lesser extent, in the I-bands; (4) a decreased heat stability of perimysial collagen indicated by a lower insoluble collagen content, lower differential scanning calorimetry transition temperature, and lower transition temperatures in isometric tension tests on muscle strips. The lactic acid injections improved significantly the textural traits of the meat (shear value, tensile strength, sensory scores) at 2 days post mortem with little further improvement when storage was extended to 14 days post mortem. Changes in texture were of similar amplitude at both post mortem injection times. The tenderization mechanisms of lactic acid injection are discussed.

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium‐dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097–2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z‐band and at the midpoint of the I‐band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I‐band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N‐terminal Z8–I5 region and the N1‐line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z‐band edge and the N1‐line as well as at the N2‐line level, two sarcomeric regions where early postmortem proteolysis is detected.

Zootechnical Performance of Cloned Cattle and Offspring: Preliminary Results

This paper presents information on the evolution of sets of cloned heifers of Holstein breed in comparison to that of control heifers derived from artificial insemination (AI) in the same farm, as well as data on a set of cloned bulls and their semen characteristics. Preliminary observations on a group of calves sired by a cloned bull and offspring of cloned females are reported. Mean birth weight in the clone group (50 females) was statistically higher than that of 68 contemporary female controls obtained by AI (49.27 +/- 10.98 vs. 40.57 +/- 5.55 kg, respectively, p < 0.05). Growth rate was within normal values for Holstein heifers (from 0.7 to 0.8 kg/day) and daily gain was not influenced by the high or low birth weight of clones. Within animals of the same clone, variability of daily gain was reduced compared to their control counterparts. Semen production from three cloned bulls was within the parameters expected for young bull of the same age. A direct comparison of morphological analysis was made between the frozen thawed semen of the donor bull and of his three clones collected at the same age. The overall semen picture appeared within acceptable limits and the clones presented similar percentages of sperm abnormalities (80% of morphologically normal spermatozoa) as the donor. These preliminary results suggest no deleterious effect of cloning on the semen picture of cloned sires. Frozen semen from one clone bull was used for an AI trial, resulting in 65% pregnancies, 25 live calves were naturally delivered. Concerning the offspring of both female and male clones, the phenotypical and clinical observation of the calves in the first week of age did not reveal any clinical abnormality, suggesting that the deviations observed in clones are not transmitted to the progeny.

Glycogen hyperaccumulation in white muscle fibres of RN- carrier pigs. A biochemical and ultrastructural study.

1. The dominant RN- gene affects meat quality of pigs by increasing the glycogen content of muscle. Glycogen localization was studied in Longissimus dorsi muscle from RN- carrier pigs (RN- pigs) and rn+ rn+ homozygous pigs (rn+ pigs). 2. Ultrastructural study showed an excess of glycogen in the sarcoplasm of white fibres from RN- pigs as compared to rn+ pigs. 3. Lysosome-enriched fractions extracted from muscles contained 6% of the tissue glycogen content in both types of pigs. The distribution of the glycogen particles between sarcoplasm and lysosomes appeared to be similar in both RN- and rn+ pig tissues. 4. White fibres from RN- pigs with an increased glycogen level showed two ultrastructural alterations: the sarcoplasmic compartment was abnormally enlarged and a large proportion of mitochondria was morphologically modified. 5. The RN- gene seems, therefore, to be associated with alterations in the glycolytic metabolism, in the distribution of proteic compartments and in the oxidative metabolism of white muscle fibres.

Effect of the RN− gene on ultrastructure and protein fractions in pig muscle

The aim of the present experiment was to study the effect of the RN(-) gene on ultrastructure of white fibres and the proportion of the different protein fractions in Longissimus lumborum muscle of RN(-) pigs. The ultrastructure of muscle was observed in 4 RN(-) carrier pigs and 4 rn(+)rn(+) homozygous pigs by mean of electron microscopy. Protein fractionation and quantification were performed on muscle from a second group of 4 RN(-) and 4 rn(+) pigs. No change in the proportions of the different protein compartments was observed. The abnormality of the glycolytic fibres of RN(-) pigs seems to be rather due to an increase of the glucid concentration in the sarcoplasm, perhaps secondarily to a modified osmolarity in the intracellular medium.