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A feeding trial was conducted to assess the effects of dietary American cockroach Periplaneta americana meal (PAM) inclusion on the growth performance, antioxidant capacity, and immunity of juvenile rainbow trout Oncorhynchus mykiss. Five isonitrogenous and isolipidic diets were formulated to contain 100, 150, 200, 250, and 300 g/kg PAM (defined as PAM100-PAM300), respectively. The final body weight and weight gain rate of PAM250 were significantly higher than those of PAM100 and PAM150, and a higher specific growth rate was recorded in PAM200-PAM300. The highest levels of liver alanine aminotransferase and serum aspartate aminotransferase (AST) as well as serum total amino acids were detected in PAM150, while higher levels of liver AST and serum total protein were detected in PAM200-PAM300. The liver lipase activity of PAM150, PAM200, and PAM300 was significantly higher than that of PAM100. The liver glucose content in PAM150 was significantly higher than that in PAM250-PAM300. The highest liver catalase, alkaline phosphatase (AKP), and lysozyme activities were detected in PAM300; the lowest liver malondialdehyde content was detected in PAM250, while the highest serum total antioxidant capacity, catalase, and total superoxide dismutase activities were detected in PAM250. The highest serum AKP and acid phosphatase activities were detected in PAM150, while higher serum IgM and complement C3 contents were detected in PAM200-PAM300. In conclusion, dietary PAM inclusion at 200-300 g/kg could improve the growth performance, antioxidant capacity, and immunity of juvenile O. mykiss.

Cloned pig fetuses produced by somatic cell nuclear transfer show a high incidence of erroneous development in the uteri of surrogate mothers. The mechanisms underlying the abnormal intrauterine development of cloned pig fetuses are poorly understood. This study aimed to explore the potential causes of the aberrant development of cloned pig fetuses. The levels of numerous fatty acids in allantoic fluid and muscle tissue were lower in cloned pig fetuses than in artificial insemination-generated pig fetuses, thereby suggesting that cloned pig fetuses underwent fatty acid deficiency. Cloned pig fetuses also displayed trophoblast hypoplasia and a reduced expression of placental fatty acid transport protein 4 (FATP4), which is the predominant FATP family member expressed in porcine placentas. This result suggested that the placental fatty acid transport functions were impaired in cloned pig fetuses, possibly causing fatty acid deficiency in cloned pig fetuses. The present study provides useful information in elucidating the mechanisms underlying the abnormal development of cloned pig fetuses.

Cloned piglets generated through somatic cell nuclear transfer (SCNT) have a high rate of neonatal death. Postnatal loss is associated with low birth weight, umbilical status and placental parameters in fertilisation-derived piglets. To investigate whether or not this relationship also exists in cloned piglets, birth weight, umbilical status, placental parameters, placental morphology and gene expression pattern were compared among four piglet groups, namely, SCNT-derived male piglets that died within 4 days (SCNT-DW4), SCNT-derived male piglets that survived over 4 days (SCNT-SO4), artificial insemination (AI)-generated male piglets that died within 4 days (AI-DW4) and AI-generated male piglets that survived over 4 days (AI-SO4). Results showed that the occurring frequency of abnormal umbilical cord in SCNT-DW4 piglets was significantly higher than that in AI-SO4 piglets but was similar to that in SCNT-SO4 and AI-DW4 piglets. The birth weight, placental surface area and placental weight of AI-SO4, AI-DW4 and SCNT-SO4 groups were similar but were significantly higher than those in SCNT-DW4 group. SCNT-SO4 placentas exhibited mild but SCNT-DW4 placentas showed severe morphological abnormalities compared with AI-SO4 placentas. The expression profiles of imprinting, angiopoiesis, nutrient transport, apoptosis and oxidative stress-related genes in SCNT-DW4 placentas were erroneous compared with those in SCNT-SO4 and AI-SO4 placentas, which both had similar gene expression patterns. These results indicate that birth weight, umbilical status, placental parameters, placental morphology and gene expression were associated with neonatal death of cloned piglets. The high loss of cloned piglets during neonatal age may be caused by severe deficiency of extra-embryonic development during prenatal stage.

Somatic cell nuclear transfer in mammalian cloning suffers from a faulty epigenetic reprogramming, which is believed to cause developmental failures in cloned embryos. Regulating the epigenetic-modifying enzymes can rescue the chromatin of cloned embryos from aberrant epigenetic status, thereby potentially promoting cloning efficiency. In this study, we investigated the effect of two histone methyltransferase inhibitors, namely, DZNep and UNC0642, on the in vitro developmental competence of cloned pig embryos. We found that (1) treatment with 10 nM DZNep or 5 nM UNC0642 for 24 h after activation had the best promoting effect on the development of cloned embryos (blastocyst rate 10.32% vs 18.08% for DZNep, and 10.44% vs 18.14% for UNC0642); (2) 10 nM DZNep and 5 nM UNC0642 significantly decreased the levels of H3K27me3 and H3K9me2, respectively, at the 2-cell, 4-cell and blastocyst stages; (3) the apoptosis level was lower in the treatment groups than in untreated control; and (4) the transcriptional expression of epigenetic genes (EZH2, GLP, G9a, Setdb1, Setdb2, Suv39h1 and Suv39h2) was decreased and pluripotency genes (Nanog, Pou5f1, Sox2 and Bmp4) was increased in treatment groups compared with control. These results indicated that treatment with DZNep and UNC0642 improves the epigenetic reprogramming of cloned embryos, which could render beneficial effect on the embryo quality and aberrant gene expression, and finally improve the developmental competence of cloned pig embryos.

Piglets cloned by somatic cell nuclear transfer (SCNT) show a high incidence of malformations and a high death rate during the perinatal period. To investigate the underlying mechanisms for abnormal development of cloned pig fetuses, we compared body weight, amniotic fluid (AF) metabolome, and placental transcriptome between SCNT- and artificial insemination (AI)-derived pig fetuses. Results showed that the body weight of SCNT pig fetuses was significantly lower than that of AI pig fetuses. The identified differential metabolites between the two groups of AF were mainly involved in bile acids and steroid hormones. The levels of all detected bile acids in SCNT AF were significantly higher than those in AI AF. The increase in the AF bile acid levels in SCNT fetuses was linked with the downregulation of placental bile acid transporter expression and the abnormal development of placental folds (PFs), both of which negatively affected the transfer of bile acids from AF across the placenta into the mother's circulation. Alteration in the AF steroid hormone levels in cloned fetuses was associated with decreased expression of enzymes responsible for steroid hormone biosynthesis in the placenta. In conclusion, cloned pig fetuses undergo abnormal intrauterine development associated with alteration of bile acid and steroid hormone levels in AF, which may be due to the poor development of PFs and the erroneous expression of bile acid transporters and enzymes responsible for steroid hormone biosynthesis in the placentas.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

Somatic cell nuclear transfer (SCNT)-derived piglets have significantly higher stillbirth rate and postnatal mortality rate than artificial insemination (AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study, stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were significantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn (SCNT-LB) piglets was significantly higher than in AI and AI-liveborn (AI-LB) piglets. Birth weight, placental weight, placental surface area and placental efficiency in SCNT and SCNT-LB piglets were significantly lower than those of AI and AI-LB piglets. Placental expression profiles of imprinting, angiopoiesis and nutrient transport-related genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.