Chinese Soft-shelled Turtle Pelodiscus Sinensis Research Articles

Antioxidant responses in hibernating Chinese soft-shelled turtle Pelodiscus sinensis hatchlings

The antioxidant defense system protects turtles from oxidative stress during hibernation. The present study examined changes of the antioxidant enzymes both on mRNA level and enzyme activity level during hibernation of Chinese soft-shelled turtle Pelodiscus sinensis hatchlings. The upstream regulator NF-E2 related factor 2 (Nrf2) mRNA was also measured. Samples were taken at pre-hibernation (17.0°C, Mud temperature (MT)), hibernation (5.8°C, MT) and arousal (20.1°C, MT). Nrf2 exhibited a tissue-specific pattern of expression with a decrease in the brain, slight increase in the liver and heart during hibernation, and significant increase during arousal in all the three tissues. Superoxide dismutase (SOD) mRNA, catalase (CAT) mRNA, and glutathione peroxidase 3 (GPx3) mRNA exhibited a similar pattern as Nrf2 in the brain and liver during the entire hibernation period. Hepatic GPx4 mRNA level increased during hibernation and decreased during arousal, whereas it did not change in the heart. Cerebral SOD and CAT activities kept stable during the experimental period, but GPx activity decreased significantly during hibernation and arousal. Hepatic GPx enzyme activity did not change, whereas those of SOD and CAT exhibited a notable decrease during arousal. Malondialdehyde concentration did not increase during the hibernation process, indicating an effective protection of the antioxidant defense system.

The effects of partial replacement of white fish meal by poultry by-product meal and addition of bile acid in feed on growth, digestibility, and serum enzyme activities of the Chinese soft-shelled turtle

Four isonitrogenous experimental diets were used to test the effects of replacing white fish meal with poultry by-product meal (PBM) and adding bile acid (BA) in a commercial feed for the Chinese soft-shelled turtle Pelodiscus sinensis with 9 replicates and 6 turtles in a 60-day study. Diet 1 contained white fish meal as a main protein source (100 %). Diet 2 contained white fish meal (60.8 %) and PBM (39.2 %) as the protein source. Diets 3 and 4 comprised the addition of 1 g kg−1 of bile acid to diets 1 and 2, respectively. Compared to diet 1, the glutamic-pyruvic transaminase level in the turtles fed with diet 2 were not augmented significantly as was the case for glutamic oxalacetic transaminase level. The relative growth rate and specific growth rate in diet 2 were the same as those of diet 1. The apparent digestibility coefficient for lipid (ADCL) tended to decrease after that white fish meal was partially replaced. The values of weight gain, relative growth rate, feeding rate, specific growth rate, apparent digestibility coefficient for dry matter, ADCL and apparent digestibility coefficient for protein (ADCP) of the turtles on diet 4 (1 g kg−1 bile acid added in diet 2) increased by 28.1, 28.8, 10.1, 20.6, 1.7, 0.6 and 0.3 %, respectively, compared to those on diet 2. The combined effects of bile acid and PBM on the growth of turtles was even more effective than the whole white fish meal diet, by increasing feeding rate 7.4 %; it decreased the amounts of crude lipids (by 22.8 %), glutamic-pyruvic transaminase (by 1.9 %), and alkaline phosphatase (by 3.9 %). Therefore, replacing 39.2 % of white fish meal with PBM and adding BA was feasible in turtle feed.

Effects of water spinach Ipomoea aquatica cultivation on water quality and performance of Chinese soft-shelled turtle Pelodiscus sinensis pond culture

The Chinese soft-shelled turtle Pelodiscus sinensis is a highly valued freshwater spe- cies cultured in China. A 122 d experiment was conducted to assess the effects of water spinach Ipomoea aquatica cultivation in floating beds on water quality, and growth performance and eco- nomic return of P. sinensis cultured in ponds. Two treatments, each in triplicate, with and without I. aquatica cultivation were designed. Results showed that the levels of total ammonia nitrogen (TAN), total nitrogen (TN), total phosphorus (TP), chlorophyll a (chl a) and turbidity in treatments with I. aquatica cultivation (IAC) were significantly (p < 0.05) lower than those in treatments with- out I. aquatica (control). Mean TN and TP concentrations in the IAC treatment were 27.9 and 42.5%, respectively, lower than in the control treatment at the end of the experiment. The pres- ence of I. aquatica also has a positive effect on the performance of P. sinensis. Although no signif- icant difference was found in specific growth rate (SGR) between the 2 treatments, mean survival rates, production and net income were significantly higher in the IAC treatment compared to the control (p < 0.05). These results suggest that I. aquatica cultivation in the pond system of turtles has a synergistic effect on overall economic return and is effective at improving turtle growth performance and water quality.

Global analysis of differential gene expression related to long-term sperm storage in oviduct of Chinese Soft-Shelled Turtle Pelodiscus sinensis.

Important evolutionary and ecological consequences arise from the ability of female turtles to store viable spermatozoa for an extended period. Although previous morphological studies have observed the localization of spermatozoa in Pelodiscus sinensis oviduct, no systematic study on the identification of genes that are involved in long-term sperm storage has been performed. In this study, the oviduct of P. sinensis at different phases (reproductive and hibernation seasons) was prepared for RNA-Seq and gene expression profiling. In total, 2,662 differentially expressed genes (DEGs) including 1,224 up- and 1,438 down-regulated genes were identified from two cDNA libraries. Functional enrichment analysis indicated that many genes were predominantly involved in the immune response, apoptosis pathway and regulation of autophagy. RT-qPCR, ELISA, western blot and IHC analyses showed that the expression profiles of mRNA and protein in selected DEGs were in consistent with results from RNA-Seq analysis. Remarkably, TUNEL analysis revealed the reduced number of apoptotic cells during sperm storage. IHC and TEM analyses found that autophagy occurred in the oviduct epithelial cells, where the spermatozoa were closely attached. The outcomes of this study provide fundamental insights into the complex sperm storage regulatory process and facilitate elucidating the mechanism of sperm storage in P. sinensis.

Expression of TLR2/4 on Epididymal Spermatozoa of the Chinese Soft-Shelled Turtle Pelodiscus sinensis During the Hibernation Season.

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods during hibernation, but the mechanism that underlies the sperm storage is poorly understood. This study was carried out to confirm the presence of TLR2/4 (Toll-like receptor 2/4) in epididymal spermatozoa during the hibernation season and to analyze whether TLRs play a role in sperm storage. The structure and ultrastructure of a spermatozoon during the hibernation stage were investigated using light- and transmission electron-microscopy. RT-PCR was used to analyze mRNA expression, while protein expression was determined via Western blot. TLR2/4 mRNA and proteins were detected in spermatozoa. Immunofluorescence staining was used to confirm TLR2/4 localization in the spermatozoon, and TLR2/4 were localized in the midpiece and the posterior segment of the head of the spermatozoon, which corresponded to the cytoplasmic droplets (CDs) of the turtle spermatozoon. As TLRs play critical roles in detecting and responding to invading pathogens, this study provided molecular evidence that TLR2/4 might contribute to sperm storage in the epididymides. Anat Rec, 299:1578-1584, 2016. © 2016 Wiley Periodicals, Inc.

Characterization and functional analysis of toll-like receptor 4 in Chinese soft-shelled turtle Pelodiscus sinensis

Mammalian Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) in initiating the innate immune responses. Early studies indicate that turtles are more resistant to LPS challenge than mammals. It remains unknown if turtles express TLR4 and why they are more resistant to LPS. In this study, TLR4 gene from Chinese soft-shelled turtle, Pelodiscus sinensis, was cloned and characterized. The full length cDNA of turtle TLR4 (tTLR4) consists of 3396 base pairs with an 2499-bp open reading frame, encoding 833 amino acids. Phylogenetic and syntenic analyses suggest that tTLR4 is to be orthologous to human TLR4. Its mRNA expression was up-regulated in spleen and blood of turtles upon Aeromonas hydrophila infection. Stimulation of turtle peripheral blood monocytes with LPS significantly upregulated tTLR4 mRNA and inflammation-related gene expression, such as Interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2). In tTLR4-expressing HEK293 cells, higher concentration of LPS exposure could enhance the activity of the NF-κB promoter, but not the INF-β promoter. Such activity required co-expression of turtle myeloid differentiation factor 2 (tMD2) and cluster of differentiation 14 (tCD14). These results provide evidence for a functional TLR4 in reptiles and, together with the syntenic analysis, support the idea that the TLR4 receptor for LPS recognition may have arisen after reptiles.

Cytological study on Sertoli cells and their interactions with germ cells during annual reproductive cycle in turtle.

Sertoli cells (SCs) play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis. This present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. During hibernation period with the quiescent of spermatogenesis, several autophagosomes were observed inside the SCs, the processes of which retracted. In early spermatogenesis, when the germ cells started to proliferate, the SCs contained numerous lipid droplets instead of autophagosomes. In late spermatogenesis, the SCs processes became very thin and contacted several round/elongated spermatids in pockets. At this time, abundant endoplasmic reticulum and numerous mitochondria were present in the SCs. The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle. Moreover, SCs were involved in the formation of cytoplasmic bridges, phagophores, and exosome secretions during spermatogenesis. Tubulobulbar complexes (TBC) were also developed by SCs around the nucleus of the spermatid at the time of spermiation. Strong, positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis. These data provide clear cytological evidence about the seasonal changes in SCs, corresponding with their different roles in germ cells within the Chinese soft‐shelled turtle Pelodiscus sinensis.

Identification and Functional Analysis of Interleukin-1β in the Chinese Soft-Shelled Turtle Pelodiscus sinensis.

Chinese soft-shelled turtle (Pelodiscus sinensis) is commercially cultured in East and Southeast Asia for its nutritional and medicinal values. In this study, we identified interleukin-1β (IL-1β) from Chinese soft-shelled turtle. The full-length cDNA of Pelodiscus sinensis IL-1β (tIL-1β) consists of 1529 base pairs with an 831-base-pair open reading frame, encoding 277 amino acids. The guanine-cytosine (GC) content in the coding sequence and 3’ untranslated region of tIL-1β is considerably higher than that of other vertebrates. Its mRNA expression level increased significantly during Aeromonas hydrophila infection. The tIL-1β lacks the typical IL-1β-converting enzyme (ICE) cut site found in mammalian IL-1β, but still could be cleaved by turtle caspase-1. By mutating the potential cleavage sites, we identified aspartic acid (Asp/D) 130 as the ICE cut site in tIL-1β. The peptide truncated at D130 was expressed using the baculovirus expression system; its bioactivity is confirmed by the ability to induce cyclooxygenase-2 (COX-2) and tIL-1β itself in peripheral blood monocytes. In conclusion, we characterized IL-1β from Chinese soft-shelled turtle and identified its D130 as a non-typical ICE cut size.

Establishment and characterization of a cell line from the Chinese soft-shelled turtle Pelodiscus sinensis.

The establishment and partial characterization of Pelodiscus sinensis continuous cell line is described here. A novel P. sinensis fibroblast cell line, designated PSF, was established from heart tissue by the semi-digestion explant culture technique. Since its initiation in July 2013, the cell line has been subcultured at 30°C in minimal essential medium (MEM) containing 15% (v/v) fetal bovine serum for more than 50 passages. The growth curve of the cell line revealed the population doubling time was 51.1h. Karyotyping analysis indicated the modal chromosome number was 66, and no microbial contamination was detected. The PSF cell line produced significant fluorescent signals after transfection with plasmid pEGFP-C3. Analysis of mitochondrial cytochrome D-loop sequences revealed 96% identity among other Chinese turtle subspecies. Several cell line characterizations included morphological analysis and immunocytochemistry, which revealed the origin of the PSF cell line was fibroblast-like cells. Measurement of the isoenzymes lactic dehydrogenase and malic dehydrogenase showed no cross-contamination of this cell line with other species. This newly established cell line will be a valuable tool for transgenic and genetic manipulation studies and will act as an efficient instrument for studies of the viral diseases of the soft-shelled turtle.

Telocytes: novel interstitial cells present in the testis parenchyma of the Chinese soft-shelled turtle Pelodiscus sinensis.

Telocytes (TCs) are novel interstitial cells that have been found in various organs, but the existence of TCs in the testes has not yet been reported. The present ultrastructural and immunohistochemical study revealed the existence of TCs and differentiate these cells from the peritubular cells (Pc) in contact with the surrounding structures in the testes. Firstly, our results confirmed the existence of two cell types surrounding seminiferous tubules; these were Pc (smooth muscle like characteristics) and TCs (as an outer layer around Pc). Telocytes and their long thin prolongations called telopodes (Tps) were detected as alternations of thin segments (podomers) and thick bead-like portions (podoms), the latter of which accommodate the mitochondria and vesicles. The spindle and irregularly shaped cell bodies were observed with small amounts of cytoplasm around them. In contrast, the processes of Pc contained abundant actin filaments with focal densities, irregular spine-like outgrowths and nuclei that exhibited irregularities similar to those of smooth muscle cells. The TCs connected with each other via homocellular and heterocellular junctions with Pc, Leydig cells and blood vessels. The Tps of the vascular TCs had bands and shed more vesicles than the other TCs. Immunohistochemistry (CD34) revealed strong positive expression within the TC cell bodies and Tps. Our data confirmed the existence and the contact of TCs with their surroundings in the testes of the Chinese soft-shelled turtle Pelodiscus sinensis, which may offer new insights for understanding the function of the testes and preventing and treating testicular disorders.

Novel cellular evidence of oviduct secretions in the Chinese soft-shelled turtle Pelodiscus sinensis.

The oviduct is the location of fertilization and sperm storage. We examined the ultrastructure of the oviduct epithelium and its glandular secretions in the isthmus, uterus and vagina of Chinese soft-shelled turtle Pelodiscus sinensis using light and transmission electron microscopy. The epithelium in these segments is lined with ciliated, secretory and other cells; the first two cell types span the entire epithelium, with secretory cells being predominant. The ciliated cells are characterized by the presence of a secretory vacuole that releases apocrine secretions into the lumen, whereas the secretory cells contain typical biphasic granules with both dark and light aspects. The third type of cells observed have wider proximal portion, abundant mitochondria, vacuoles, and narrow nuclei. The storage of spermatozoa is restricted to the isthmus, uterus, and vagina. In addition, the gland cells show prominent features, including the presence of granules of different shapes, sizes, and electron densities. The synthesis of these granules is described for the first time in this study. Mitochondria appear to play an important role in the formation of dense granules, the rough endoplasmic reticulum and microfilaments may also play a role in the maturation of these dense granules. After completing the maturation process, these granules are released into the lumen of the gland cells.

Identification and validation of novel microrna molecule from the Pelodiscus sinensis by bioinformatics approaches

MicroRNAs (miRNAs) are a group of endogenous, short non-coding RNAs with the length of about 22 nt, which mediate gene expression at the post-transcriptional level through mRNA degradation or translational repression. Among them, some are highly evolutionally conserved in the animal kingdom; this provides a powerful strategy for identifying miRNAs in new species. The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important freshwater aquaculture reptilian species in China, but related miRNAs have not been identified up to now. In the present study, a total of 10 Pelodiscus sinensis miRNAs were identified according to Pelodiscus sinensis ESTs and GSSs information in NCBI database by bioinformatics approaches. The RT-PCR-based assays were performed and found that 10 Pelodiscus sinensis miRNAs were expressed. Using these miRNAs, 22 target genes were identified. These genes encode 22 proteins involved in metabolism, signal transduction, transcriptional regulation, and development. These miRNAs and their targets will serve as useful resources for their, functional analyses in miRNA-regulated processes in Pelodiscus sinensis breeding and genetic research.

Effects of fish meal replacement with animal protein blend on growth performance, nutrient digestibility and body composition of juvenile Chinese soft-shelled turtlePelodiscus sinensis

An 8-week feeding trial was conducted to evaluate the potential of partial replacement of fish meal with rendered animal protein blend (APB) [meat and bone meal (MBM): expanding blood meal (EBM) = 4 : 1] in the diet of juvenile Chinese soft-shelled turtle Pelodiscus sinensis. Seven isonitrogenous and isoenergetic practical diets replacing 0%, 10%, 20%, 30%, 40%, 50% and 60% fish meal protein by APB were formulated. Weight gain, feed efficiency, protein efficiency and the apparent digestibility coefficients (ADCs) of dry matter and gross energy in the experimental diets all exhibited no significant difference (P > 0.05) when dietary fish meal was replaced for 0–40%, but decreased significantly (P 0.05) among all the treatments. Nitrogen retention and the ADCs of protein and most amino acids all decreased significantly (P < 0.05) when fish meal was substituted by more than 30%. The results indicated that fish meal in the practical diet of juvenile Chinese soft-shelled turtle could be replaced by 30–40% using a combination of MBM and EBM without negative effects on growth performance, nutrients digestibility and body composition.

Identification and characterization of telocytes in the uterus of the oviduct in the Chinese soft-shelled turtle, Pelodiscus sinensis: TEM evidence.

Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead-like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft-shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft-shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.

Development and characterization of 17 microsatellite markers for Chinese soft-shelled turtle, Pelodiscus sinensis

A set of 17 polymorphic microsatellite markers were isolated and characterized for the Chinese soft-shelled turtle (Pelodiscus sinensis) using a next generation sequencing approach. The number of alleles per locus ranged from 2 to 11. The observed, expected heterozygosities and polymorphic information content per locus varied from 0.103 to 0.750, 0.222 to 0.883 and 0.158 to 0.788, respectively. Significant linkage disequilibrium and deviations from the Hardy–Weinberg disequilibrium between two pairs of loci were found. These loci will be beneficial for the population genetic structure investigation and marker assisted breeding for P. sinensis.

A common developmental plan for neocortical gene-expressing neurons in the pallium of the domestic chicken Gallus gallus domesticus and the Chinese softshell turtle Pelodiscus sinensis.

The six-layered neocortex is a unique characteristic of mammals and likely provides the neural basis of their sophisticated cognitive abilities. Although all mammalian species share the layered structure of the neocortex, the sauropsids exhibit an entirely different cytoarchitecture of the corresponding pallial region. Our previous gene expression study revealed that the chicken pallium possesses neural subtypes that express orthologs of layer-specific genes of the mammalian neocortex. To understand the evolutionary steps leading toward animal group-specific neuronal arrangements in the pallium in the course of amniote diversification, we examined expression patterns of the same orthologs and a few additional genes in the pallial development of the Chinese softshell turtle Pelodiscus sinensis, and compared these patterns to those of the chicken. Our analyses highlighted similarities in neuronal arrangements between the two species; the mammalian layer 5 marker orthologs are expressed in the medial domain and the layer 2/3 marker orthologs are expressed in the lateral domain in the pallia of both species. We hypothesize that the mediolateral arrangement of the neocortical layer-specific gene-expressing neurons originated in their common ancestor and is conserved among all sauropsid groups, whereas the neuronal arrangement within the pallium could have highly diversified independently in the mammalian lineage.

IFN-γ in turtle: Conservation in sequence and signalling and role in inhibiting iridovirus replication in Chinese soft-shelled turtle Pelodiscus sinensis

The IFN-γ gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-γ family signature motif 130IQRKAVNELFPT, an NLS motif 155KRKR and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-γ at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-γ in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-γ in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-γ may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation.

Determination of Residues of Malachite Green and Leucomalachite Green in Chinese Soft-Shelled Turtle (Pelodiscus sinensis) Muscle by Ultra-High Performance Liquid Chromatography-Mass Spectrometry

A method for the determination of residues of malachite green and leucomalachite green in Chinese soft-shelled turtles (Pelodiscus sinensis) muscle by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The samples extracted with acetonitrile, and clean up on a neutral alumina SPE cartridge, separated on a C18 column using acetonitrile-5mmol/L ammonium acetate as mobile phase, and then detected under multiple reaction monitoring mode.The method showed a good linearity for all the analysts over the range of 0.1-50μg/L with r20.999.The detection limits of malachite green and leucomalachite green were 0.10 and 0.12μg/kg respectively. The recoveries ranged from 84.9 % to 93.8% at three spiked concentrations with the relative standard deviations lower than 5%. The results shows that this method has the advantages of easy to operate, fast to perform,with high sensitivity and accuracy, and it is suitable for detection of residues of malachite green and leucomalachite green in Chinese soft-shelled turtle (Pelodiscus sinensis).

Morphologic analysis of atretic follicles in the Chinese soft-shelled turtle, Pelodiscus sinensis

The Chinese soft-shelled turtle (Pelodiscus sinensis) and its eggs have been utilized as both a source of nutrition and medicine for thousands of years. In the present study, follicular atresia was investigated by morphological analysis as an important factor affecting clutch size. Results showed that follicular atresia can be divided into four stages: (1) development of follicular cell disorder; (2) massive cellular proliferation in the ooplasm with elimination of yolk granules; (3) large amounts of erythrocytes appearing in the ooplasm; (4) gradual degeneration of atretic follicles. For the first time, we have shown follicular atresia to be a dynamic process in P. sinensis and found that yolk granules are eliminated by both liquefaction as well as non-liquefaction processes. The present study may provide detailed morphologic analysis for further research into egg quality and clutch size in P. sinensis.

Do fluctuations in incubation temperature affect hatchling quality in the Chinese soft-shelled turtle Pelodiscus sinensis?

The Chinese soft-shelled turtle Pelodiscus sinensis is the most common turtle species sold in China. In most turtle farms P. sinensis eggs are incubated at fluctuating temperatures. To examine if fluctuations in incubation temperature affect hatching success, incubation length and hatchling quality, we incubated P. sinensis eggs in five incubators: one set at 30°C, two ramp-programmed at 30±3°C and±5°C; the remaining two step-programmed at 30±3°C and±5°C. Temperatures changed at 12h (+) and 12h (−) in the ramp-programmed incubators, and at 6h intervals in an order of lowest–mean–highest–mean levels in the step-programmed incubators. Size-related (carapace length, carapace width and mass) rather than performance-related (swimming speed and early growth) hatchling phenotypes differed among the five treatments. Eggs in ramp-programmed incubators overall produced larger hatchlings than did those in step-programmed incubators, and eggs at 30°C produced larger hatchlings than did those incubated at fluctuating temperatures. However, temperature-induced variation in hatchling size was not a significant source of variation in post-hatching growth, and the pattern of fluctuations in incubation temperature was less important for turtles reared in the laboratory. We suggest that the most important thing that should be done by farmers is to avoid exposure of P. sinensis eggs to the temperatures potentially lethal to embryos rather than to incubate them at stable temperatures.