Rapidly developing and intensive animal husbandry in the livestock industry is a major contributor to global environmental pollution [1,2]. Phytase, an enzyme that breaks down indigestible phytic acid into inorganic phosphorus, cannot be secreted by monogastric animals such as pigs and chickens [3]. Large quantities of manure waste containing phytate phosphorus are generated by livestock, leading to environmental pollution [1,4]. Therefore, it is important to economically utilize phosphorus resources and protect the environment by improving the utilization rate of phytic acid phosphorus in feed [5]. Phytase is a kind of hydrolase that can decompose phytic acid into inositol and phosphoric acid, which can be digested and absorbed by animals [[6], [7], [8]]. Phytase can effectively increase phytic acid phosphorus utilization in the diet and decrease fecal phosphorus emissions, reducing environmental phosphorus pollution [9].

Handmade cloning (HMC), a simplified alternative to micromanipulation based on the traditional cloning nuclear transfer (NT) technique, has been developed in multiple phases in recent years [[10], [11], [12]]. HMC is a low cost, high efficiency zona-free NT technique that does no requirement micromanipulation [13,14]. Booth et al. [15] first obtained embryo cell cloned bovines using HMC in 2001, and Du et al. [10] generated the first pigs using HMC in 2007. In the present study, HMC was used to generate cleavage-resistant transgenic pigs.

The development of genetic engineering technology, especially the improvement of phytase production in transgenic animals, has provided an effective new approach to solve these problems. Using genetic engineering, endogenous phytase with high biological activity has been produced in the digestive tract of monogastric animals such as pigs and poultry [16]. In 2001, transgenic mice with the phytase gene were generated by Golovan et al. [17], and enviropig with the same phytase gene were made as well [18], significantly reducing phosphorus content in pig feces. Yin et al.'s phytase transgenic models, induced via microinjection, showed significantly reducing phosphorus in pig feces [19]. Furthermore, the exogenous phytase gene was identified to stably transmit and express in the salivary glands of domestic food animals [20]. Nevertheless, the production of cleavage-resistant phytase, which is insensitive to cleavage by pepsin and trypsin, has not been reported. Using this type of phytase could offer more time for phytases to work with the substrate and meet the production requirement of endogenous enzymes in transgenic animals.

The present study constructed a vector with the cleavage-resistant phytase gene, which is insensitive to cleavage by pepsin and trypsin and has a higher affinity for the substrate with the pig parotid secretory protein (PSP) gene promoter. Using this modified phytase gene, cleavage-resistant phaytase transgenic pigs were successfully generated, offering a unique biology approach to managing phosphorus nutrition and environmental pollution in animal husbandry.