65 RARE AND OFTEN UNRECOGNIZED CEREBROMENINGITIS AND HEPATOPNEUMONIC CONGESTION ARE MAJOR CAUSES OF SUDDEN DEATH IN CLONED MALE PIGLETS

In this study, we investigated the relationship between the sex of cloned pigs and sudden infant death syndrome (SIDS). Three cell lines (2 male and 1 female) were obtained from F1 fetuses derived from 3 different dams (Yorkshire) inseminated by the same sire (Landrace); one female fibroblast cell line was obtained from a Duroc-strain fetus acquired from a slaughterhouse, the age of the fetus unknown. The fetal fibroblast cells were cultured in DMEM supplemented with 10% fetal bovine serum under 5% CO2 in air at 37°C. For NT, we used the 4 cell lines described above. All 37 cloned piglets derived from 10 pregnant recipients were alive at term and began breathing readily. Of those piglets born, 18/22 males and 5/15 females died within 2 months of age. A total of 350 paraffin blocks from 18 deceased cloned male piglets and 90 paraffin blocks from six age-matched normal control piglets were prepared from the midbrain, medulla oblongata, liver, lung, kidney, spleen, small and large intestine, thymus, uterus, placenta, ovary, testis, skin, and skeletal muscle. We found that the birth weights of male clones were 57% lower than those of control age- and sex-matched piglets. Piglets with low birth weight (<900 g) had more than twice the risk of SIDS relative to those piglets weighing more than 1000 g. The low birth weights of the cloned male piglets (0.84 ± 0.05 kg) were not merely an artifact, as the average birth weights of cloned female piglets (1.47 ± 0.09 kg) were not lower than weights of piglets produced by AI-derived control female piglets (1.36 ± 0.12 kg). An initial examination of brain samples from 18 cloned male piglets that died soon after birth identified seven piglets with meningitis characterized by severe neutrophilic inflammation in the temporal brain lobes (38.8%, 7/18). We verified meningitis when more than 1000 neutrophils were counted per cubic millimeter of tissue. Next, we found hepatopneumonic congestion (16.6%, 3/18). The deceased male clones with meningitis showed extensive neuronal cell death and blood-brain barrier damage, whereas cloned piglets with congestion had fewer and larger alveolar air sacs. Extensive alveolar cell death, especially of pneumocytes and the bronchial epithelium, was confirmed by TUNEL assay. Lung and liver congestion may be caused by a slowly flowing blood stream from heart, resulting in CO2 and O2 exchange problems. Although the gross anatomy of the cloned male piglets was normal, they were associated with other severe handicaps, the commonest being leg abnormality which occurred in 33% (6/18) of dead male cloned piglets followed by Leydig cell hypoplasia and short face. Even though 4 of 41 AI-derived control piglets died within 1 week after birth, we could not find any anomalies in them. Thus, the present study suggests that our data might reflect sex difference but not cell type difference, and that death of the cloned male piglets might be caused by risk factors of sudden infant death syndrome such as low birth weight and multiple organ failure in conjunction with hepatopneumonic congestion and cerebromeningitis.

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