# Effects of Roughage on the Lipid and Volatile-Organic-Compound Profiles of Donkey Milk
## 1 Introduction  
Dairy products are among the most common foods in daily life, accounting for 14% of global agricultural trade [1; 2]. With ongoing improvements to quality of life, demand for high-quality and diverse dairy products continues to increase. Donkey milk has been demonstrated to be a nutritional foodstuff for the human diet owing to its content of bioactive chemicals, specifically lipids, that either directly or indirectly modulate the intestinal environment's immunity, influence glucose and lipid metabolism, and lower fat buildup in skeletal muscle [3; 4; 5]. Milk lipids are not only an important indication of milk quality, they also play an important role in promoting infant brain development, regulate the cell cycle and calcium absorption, reduce inflammatory factors, and reduce skeletal muscle lipid accumulation [6; 7; 8; 9; 10]. Milk typically contains 98% triacylglycerglycerols (TGs), 1% phospholipids (PLs), and 0.3-0.5% cholesterols [9; 11]. Donkey milk has a low fat content (0.3-1.8 g/100 g), yet it contains up to 30.7% essential fatty acids (FAs) (e.g., linoleic and linoleic acids), which is 25% higher than cow's milk [5; 12]. Therefore, donkey milk is an emerging choice for people seeking a healthy diet. Previous LC-MS studies have identified 335 lipids from 13 subclasses in donkey milk, including TGs, phosphatidic acids (PAs), phosphatidylcholines (PCs), and phosphatidylethanolamines (PEs) [13]. In addition, the volatile organic compounds (VOCs) in milk are vital indicators by which consumers evaluate the quality of milk and decidewhether to purchase [14]. The basic VOCs in milk have been identified as ketones, aldehydes, methyl acids, short-chain alcohols, lactones, pyrazines, and thiols [15; 16], while a GC-MS study found that 2-heptanone, 1,3-bis-(1,1-dimethylethyl)-benzene, nonanal, D-limonene, octanoic acid, and 1-octanol are the characteristic VOCs in fresh donkey milk [17]. The lipids and VOCs in milk are influenced by several factors, including metabolic differences in the animal, feed ingredients and additives, biological enzymes, microorganisms, and production processes [18; 19]. Importantly, several studies have demonstrated that diet is the most important factor affecting milk lipid and VOC profiles [18; 20; 21; 22]. The quality of the roughage affects the energy content of the feed and accounts for 50%-80% of a donkey's diet [23]. Many studies have demonstrated that high-quality roughage (herbage, crop straw) increases an animal's dry-matter intake and affects FA uptake by the mammary glands, which in turn increases milk production, milk protein content, and milk lipid content, resulting in an altered milk lipid profile [24; 25]. Roughage's influence on microbial fermentation and hydrogenation modifies the composition of milk. Specifically, animals inhale VOCs from roughage, which then enter the bloodstream and the milk [18; 26]. A previous study revealed that milk from cows fed grass-clover silage accumulates more lipid hydroperoxides and hexanal than milk from cows fed hay [27]. Higher levels of \(\alpha\)-linolenic acid and vaccenic acid as well as higher milk yields have been reported for sheep fed hempseed cake than those fed hemp seed [28]. Another study revealed that sheep fed on New Zealand pasture showed a significant increase in \(\gamma\)-12:2 lactone content [22], while Italian forage significantly increased the content of active aldehydes, esters, and terpenoids in cheese VOCs [29]. These studies have shown that VOCs in milk can be modified through feed modification. However, there are surprisingly few studies on the effects of dietary roughages on milk quality, especially in terms of the milk lipids and VOCs in donkey milk. LC-MS and GC-MS are simple and sensitive techniques with high separation capacity and a wide analytical range that are commonly used for the analysis of milk components [30; 31; 32]. Therefore, the present study aimed to investigate the effects of different dietary roughages on the lipid and VOC profiles of donkey milk using LC-MS and GC-MS.