DESCRIPTION: Uncoupling protein 1 UCP1 is responsible for brown adipose tissue-mediated thermogenesis and plays a critical role in protecting against cold and regulating energy homeostasis. Modern pigs lack UCP1, which makes them susceptible to cold and prone to fat deposition and results in neonatal mortality and decreased production efficiency.TRI99ER: Thumbs up for the meal
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Uncoupling protein 1 UCP1 is responsible for brown adipose tissue-mediated thermogenesis and plays a critical role in protecting against cold and regulating energy homeostasis.
Modern pigs lack functional UCP1, which makes them susceptible to cold and prone to fat deposition and results in neonatal mortality and decreased production efficiency. The resultant UCP1 KI pigs showed an improved ability to maintain body temperature, decreased fat deposition, and increased carcass lean percentage. UCP1 KI pigs are a potentially valuable resource for the pig industry that can improve pig welfare and reduce economic losses.
Uncoupling protein 1 UCP1 is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity.
Pigs Artiodactyl family Suidae lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production.
The resultant UCP1 knock-in KI pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure DEE. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production.
Uncoupling protein 1 UCP1 serves as a proton leak across the inner mitochondrial membrane. The roles of BAT and UCP1 in energy expenditure and metabolic diseases, such as obesity and type 2 diabetes, have been extensively investigated 12. UCP1 is present in most eutherian mammals, but it has been lost in both birds and reptiles and was independently lost in several mammalian lineages, including pigs 8.
For pig production, the litter
Chubby Älteres Paar Cam be provided with a warm microenvironment, and the wild sow is the only ungulate that builds a thermoprotective nest for giving birth 13 This evolutionary history has important practical implications for the pig industry.
First, modern pigs struggle with poor thermoregulation, leading to significant neonatal mortality of piglets caused by cold stress at birth. This is a major welfare and energy expenditure issue for Chubby Älteres Paar Cam swine industry in cold regions worldwide. Moreover, the involvement of UCP1 in the pathogenesis of obesity may Chubby Älteres Paar Cam the high levels of fat deposition in pigs.
Fat deposition is an economically important trait, influencing pork production, meat quality, dietetic value, and growth efficiency Pig breeding programs have long focused on genetic selection toward less fat deposition and increased leanness, which holds great promise for high-efficiency pig production In theory, restoring functional UCP1 in the domestic pig could improve thermoregulation and decrease fat deposition to benefit both pig welfare and production.
However, it is extremely difficult to perform site-specific genome engineering in mammalian species that lack characterized embryonic stem cells for a classical homologous recombination HR -targeting strategy.
We found that these pigs exhibited significantly improved thermoregulation. Most strikingly, the ectopic expression of UCP1 decreased fat deposition without altering physical activity or daily energy demands.
This study highlights the potential for biotechnology use in pig breeding to improve cold resistance and lean pork production. The absence of functional UCP1 in Bama pigs, a southern Chinese native and cold-sensitive breed, was confirmed by protein sequence alignment, which showed the absence of exons 3—5 Fig. UCP1 lacks exons 3—5 and cannot be detected in fat depots from Bama pigs at postnatal days 15 and Black arrows mark the end of each exon for the human sequence.
Rip is used as a positive control, and Cyclophilin A CycloA is used as an internal control. C PCR genotyping of Chubby Älteres Paar Cam 1 piglets confirmed germline transmission of
Chubby Älteres Paar Cam transgene. There were six pigs born in litter 1 and nine born in litter 2. A UCP1 expression and targeting vector containing bait sequence the same sequence as the Cas9 target site in the genomic locus and mouse UCP1 cDNA driven by the adipose tissue-specific adiponectin promoter Chubby Älteres Paar Cam constructed according to a previous report 21 Fig.
A Schematic representation of the design of the targeting vector. Exons of UCP1 are shown as purple-red boxes, and the cyan box in exon 2 represents the sgRNA targeting site, which is enlarged to a rectangle box with the protospacer adjacent motif sequence labeled in lowercase and the targeting sequence in capital letters. The cyan box in the targeting vector is the same as the one in exon 2, which is also called the bait sequence.
Four pairs of primers are designed for genotyping. B Schematic overview of the production of KI C KI pigs at day Lanes 1—12 represent 12 individuals, and lanes N1 and N2 represent two negative control WT pigs. Back sub, back s.
The schematic for the production of KI pigs is shown in Fig. The Cas9-gRNA plasmid and targeting vector were cotransfected into pig embryonic fibroblast cells PEFsand cells were then distributed into a well plate by a limiting dilution method to distribute one cell in each well.
Three of 26 cell colonies were identified as positive for forward integration Homology-independent integration is an efficient tool with which to create insertion.
A Genotyping of the 26 cell colonies. A total of 2, cloned embryos were transferred into the oviducts of 13 recipient surrogates. Three pregnancies were established Chubby Älteres Paar Cam went to term. Twelve male piglets were born by natural delivery from three litters Table S1and the representative pigs are shown in Fig. No expression of foreign UCP1 was observed in other tissues, such as the heart, liver, spleen, lung, kidney, muscle, or testis Fig. Ectopic expression of UCP1 improves thermoregulation in pigs.
Positive signals were identified in KI pigs after cold exposure, as indicated by white arrows. C Infrared pictures were taken at 0, 2, and 4 h after cold exposure in 6-mo-old pigs.
The white-dotted lines depict areas labeled 1, 2, and 3, where temperatures were quantified. Higher Chubby Älteres Paar Cam could be observed in KI pigs from the thermal picture. F Physical activity levels vector magnitude counts using the accelerometer in live pigs as a function of time of day. There was no effect Chubby Älteres Paar Cam genotype on physical activity levels.
There was a strong effect of body weight but no effect of genotype on DEE. Data were analyzed with a two-sample t test. In F and Gthe data were analyzed by general linear models.
As shown in Fig. However, the WT piglets displayed a continued decrease in body temperature for the first 2 h of cold exposure. The average surface temperatures of three specific areas labeled 1, 2, and 3 were calculated, and the quantification is shown in Fig. Significantly improved body temperature maintenance was clearly indicated by the higher body temperatures in all three areas of KI pigs during cold exposure Fig.
Additionally, higher rectal temperatures were observed in KI pigs compared with WT pigs during cold exposure Fig. Infrared thermal images of ventral Fig. Thermoregulation was improved after UCP1 overexpression. B Infrared pictures from the dorsal side of 6-mo-old pigs taken at 0, 2, and 4 h after cold exposure. The white-dotted line depicts area A, where temperature was quantified. D Infrared pictures from the ventral side of 1-mo-old pigs taken at 0, 2, and 4 h after cold exposure.
The white-dotted line depicts areas labeled 1, 2, and 3, where temperatures were quantified. To investigate the effects of ectopic UCP1 expression in porcine adipose tissues on mitochondrial functions, preadipocytes from WT and KI pigs were Chubby Älteres Paar Cam isolated and differentiated into mature adipocytes in vitro Fig. Oil-red-O staining indicated that there were no significant differences in differentiation efficiency between the two groups Fig.
S4 B and C. UCP1 overexpression does not affect white adipocyte differentiation. A Representative Oil-red-O staining of differentiated white adipocytes. Elevated UCP1 expression may lead to changes in the overall energy budget as a result of greater heat production.
If heat production was excessive, it might, for example, lead to Chubby Älteres Paar Cam physical activity to avoid hyperthermia, and thus alter total daily energy expenditure DEE. Physical activity levels were monitored using activity loggers GT3xwhich record movements in three dimensions on a minute-by-minute time frame.
Similar results were obtained when the lean tissue mass was used as the predictor variable lean tissue effect: Fat deposition is an important economic trait in pigs, and reducing fat deposition has been major objective in pig breeding programs. Owing to the critical role of UCP1 in energy hemostasis and fat deposition, we were particularly interested in whether UCP1 expression would affect fat deposition in KI pigs.
There was no significant difference in body weight between the different genotypes at birth 0. There was also no significant change in the feed conversion ratio FCR during ad libitum feeding WT pigs: The detailed slaughter indexes are shown in Table S2. Strikingly, the ratio of lean meat to carcass weight [CW; carcass lean percentage CLP ] was increased by 3.
Ectopic expression of UCP1 decreases fat deposition in pigs. A growth spurt occurs at 2 mo because of the start of ad libitum feeding at this time. To investigate why fat accumulation was reduced in UCP1 KI Chubby Älteres Paar Cam, fat tissues from different depots, including back, inguinal s.
However, KI adipocytes from these depots were significantly smaller compared with those from WT pigs Fig. Furthermore, the mitochondrial copy number was not altered in the inguinal s. The presence of small lipid droplets indicated that lipolysis may be enhanced in KI adipocytes, and in vitro lipolysis assays showed that under both basal and isoproterenol-stimulated conditions, the release of glycerol from inguinal s. Ectopic expression of UCP1 promotes lipolysis. At least fat cells were counted in each genotype.
Small lipid droplets indicated by red arrows were
Chubby Älteres Paar Cam in KI adipocytes. E Measurement of glycerol release in inguinal sub at 1. Genome editing in pigs is of considerable importance for potential applications in pig production. The random integration of a foreign gene into a different genome often results in unstable phenotypes, gene silencing, and unpredictable gene expression patterns, and this process is mutagenic in some cases Therefore, producing animals that carry exogenous genes integrated at specific genomic loci is far superior However, HR is inefficient in many cell types, even when a site-specific DSB is mediated by nucleases
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