Simple Summary
Canine and feline milk are vital for the early development of offspring, yet their lipid profiles differ significantly from the bovine and caprine milk commonly used in milk replacers. This study systematically compared the lipid composition of milk from dogs, cats, cows, and goats. The results showed that the concentrations of total lipids and phospholipids in canine and feline milk were significantly higher than those in bovine and caprine milk. Additionally, canine and feline milk contained higher levels of unsaturated fatty acids and were rich in docosahexaenoic acid and arachidonic acid. Notably, we also observed that a large proportion of palmitic acid was located at the sn−2 position of triacylglycerols in canine and feline milk, a structural feature that may influence digestion. Through lipidomic analysis, more than 2700 lipid molecules were identified, and several specific molecules that can serve as characteristic markers for canine and feline milk were discovered. These findings provide key scientific evidence for the precise development of specialized milk replacers that meet the nutritional needs of puppies and kittens, supporting their healthy growth.
Keywords: canine milk, feline milk, fatty acid positional distribution, triacylglycerol, lipidomics
Abstract
This study systematically compared the lipidomes of canine, feline, bovine, and caprine milk. Feline milk contained the highest total lipid content (110.83 mg/mL), significantly exceeding that of canine (81.52 mg/mL), caprine (40.27 mg/mL), and bovine milk (36.25 mg/mL). The phospholipid content in both canine (0.97 mg/mL) and feline milk (0.90 mg/mL) was approximately three times higher than that in bovine and caprine milk (approximately 0.30 mg/mL). Compared to bovine and caprine milk (approximately 30%), canine and feline milk had markedly higher proportions of unsaturated fatty acids (approximately 70%) and were enriched with functional long-chain polyunsaturated fatty acids, including arachidonic acid and docosahexaenoic acid. A distinctive feature was that over 60% of palmitic acid was esterified at the sn−2 position of triacylglycerols in canine and feline milk, a structural similarity shared with human milk. Lipidomic analysis identified 2708 lipid molecules across the four milk types, revealing several triacylglycerol species as potential species-specific biomarkers. These findings provide a concrete scientific basis for developing precisely formulated milk replacers that meet the specific nutritional requirements of puppies and kittens.
1. Introduction
As crucial companion animals worldwide, dogs and cats depend heavily on early-life nutrition for healthy development. The neonatal period represents a critical transitional phase, characterized by rapid adaptation to the extrauterine environment and preparing them for subsequent weaning [1]. Nutrition at this phase impacts immediate survival and health while also shaping long-term growth and metabolic programming [2]. As the ideal nutritional source for puppies and kittens, milk not only supplies energy but also provides immune-protective components, vital nutrients, and bioactive compounds that are essential for the development of their nervous systems and cognitive abilities [3]. However, rising pet populations, diverse rearing conditions, and the growing number of stray animals have led to situations where many newborns cannot obtain sufficient maternal milk, resulting in malnutrition and increased health risks. In this context, commercial milk replacers have become essential substitutes, widely used for orphaned, weak, or diseased neonates, as well as for supplementing large litters and supporting the weaning transition [4].
Although commercial milk replacers have been used for decades, their typical ingredients, such as bovine or caprine milk, differ significantly in composition from canine and feline milk [5]. Studies indicate that the lipid content in canine milk ranges from 8.92% to 14.31%, while in feline milk it is approximately 12.7%; both levels are substantially higher than the 3–5% found in bovine or caprine milk. Furthermore, the fatty acid (FA) profiles of total lipids in canine and feline milk are notably distinct from those in bovine and caprine milk, characterized by a higher proportion of unsaturated fatty acids (UFAs) [6,7]. Current research remains largely focused on total lipid content and basic FA composition, with insufficient systematic lipidomic characterization, particularly regarding lipid molecular species composition and fatty acid distribution. This gap limits the ability of milk replacers to accurately mimic the unique lipid architecture of canine and feline milk. Therefore, a systematic analysis of their lipid profiles is of critical theoretical importance for developing species-specific milk replacers and stage-specific feeds.
Milk fat constitutes a highly complex natural lipid system and serves as a critical source of energy and nutrients [8]. It provides approximately 40–50% of the dietary energy intake to offspring, while also delivering essential FAs, fat-soluble vitamins, and a range of bioactive compounds that profoundly influence growth [9]. The lipids in milk are organized into globules comprising a triacylglycerol-rich core surrounded by a tri-layer membrane, designated as the milk fat globule membrane (MFGM) [10]. It is well established that the functional and nutritional properties of triacylglycerols (TAGs), the primary constituents of milk fat, are governed by both their FA composition and the stereospecific position of these FAs on the glycerol backbone [11]. Saturated fats, especially palmitic acid (C16:0), are preferentially esterified at the sn−2 position of TAGs, while unsaturated fats like oleic acid (C18:1n−9) and linoleic acid (C18:2n−6) are enriched in the sn−1,3 positions [12]. Evidence from human infant nutrition and model systems suggests that higher sn−2 palmitate may support calcium and fat absorption, bone mineralization, and the growth of beneficial gut microbiota [13]. This specific structural arrangement is exemplified by the representative structured triacylglycerol—1,3-dioleoyl-2-palmitoyl-glycerol (OPO) [14]. Although present at low levels, phospholipids (PLs) have attracted considerable focus for their involvement in constructing the MFGM and their diverse bioactive properties. Accumulating evidence indicates that dairy-derived PLs play integral roles in promoting infant and child health by supporting immune function, brain development, and intestinal homeostasis [15]. Currently, the lipid compositions of human, bovine, and caprine milk have been relatively well-established [16,17]. However, systematic analyses of lipid profiles in canine and feline milk remain limited, highlighting a gap that warrants further in-depth investigation.
Therefore, the primary objective of this study was to conduct a comparative lipid analysis of canine, feline, bovine, and caprine milk, with a specific focus on: (i) quantifying total lipid and PL contents; (ii) characterizing FA composition; (iii) determining the positional distribution of fatty acids at the sn−2 position of TAGs; and (iv) comprehensively comparing lipid molecular profiles and systematically screening for significantly different lipids (SDLs) and potential lipid biomarkers based on multi-level statistical models. The findings offer a theoretical foundation and data critical for formulating nutritionally adequate milk replacers and early-life stage feeds tailored for canines and felines.
2. Materials and Methods
2.1. Chemicals and Reagents
Mass spectrometry-grade solvents, including acetonitrile, isopropanol, and n-hexane, were obtained from Fisher Scientific (Waltham, MA, USA), while mass spectrometry-grade formic acid and ammonium formate were procured from Sigma-Aldrich (Shanghai, China). The source of all other analytical-grade solvents and reagents was Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Milk Sample Collection
Milk samples were collected from four species, with four individuals per species: Shiba Inu dogs, American Shorthair cats, Guanzhong goats, and Holstein cows. Canine and feline milk samples were obtained from healthy individuals (approximately 2 years old) at 3–4 weeks postpartum, provided by the Gambol Pet Group Co., Ltd. (Liaocheng, China). Fresh caprine and bovine milk were purchased from local farms in Qingdao, China. A minimum volume of 5 mL was collected per individual. All samples were transported on ice to the laboratory and stored at −80 °C until analysis, usually within 2 weeks of collection.
2.3. Lipid Extraction
Lipids from the milk samples were extracted following the Bligh and Dyer method [18]. Briefly, following a 30 min incubation at 40 °C in a chloroform/methanol mixture (1:2, v/v), the 0.5 mL milk samples were subjected to centrifugation at 6000× g for 10 min at 4 °C. After three repetitions of this extraction, the pooled chloroform layers were concentrated under reduced pressure to yield the total lipids.
2.4. Total Lipid Quantification
The lipid content in milk was determined gravimetrically [19]. Briefly, an aliquot of milk sample was subjected to lipid extraction, and the organic phase was then rotary-evaporated to constant weight. The total lipid content was calculated as the ratio of the lipid mass after evaporation to the volume of the initial milk sample.
2.5. Phospholipid Quantification
Phospholipid (PL) content was quantified by the ammonium molybdate method [20]. A standard curve was constructed using sodium dihydrogen phosphate, which demonstrated a highly linear relationship between phosphorus content (x) and absorbance (y): y = 0.1874x–0.0102 (R2 = 0.9998). Then, 5 mg of milk fat was digested with 0.5 mL of perchloric acid at 160 °C until the solution became colorless. Then, 3.5 mL of water and 1 mL of an ammonium molybdate/ascorbic acid (VC) mixture (1:1, v/v) were added. Its absorbance was measured at 820 nm. The PL content (X, mg/mL) was determined according to the following Formula (1):
| X = (P × V1 × 25)/(V× V2) | (1) |
where P is the phosphorus content of the test solution (mg) derived from the standard curve; V denotes the volume of the test portion (mL); V1 represents the fixed total volume of the sample test solution (mL); V2 denotes the volume of the sample solution employed for the analysis; 25 is the conversion factor, representing the milligrams of phospholipids equivalent to 1 mg of phosphorus.
2.6. Fatty Acid Analysis
2.6.1. Analysis of Total Lipid Fatty Acid Composition
FA composition was determined using a previously reported method [18]. Briefly, 2 mg of lipid was mixed with 2 mL of HCl/methanol (1:5, v/v) in a sealed vial and incubated at 90 °C for 3 h to convert FAs to methyl esters. The methyl esters were analyzed using an Agilent 7820 gas chromatograph (GC) equipped with a flame ionization detector. Separation was achieved on a Supelcowax quartz capillary column (30 m × 0.32 mm × 0.25 μm). The oven temperature program was set as follows: initial hold at 170 °C for 5 min, ramped to 240 °C at 5 °C/min, and finally held at 240 °C for 50 min. FAs in the samples were identified based on comparison with the retention times of a standard methyl ester mixture. Quantification was performed using the area normalization method, with results expressed as the relative percentage of each FA to the total FAs.
2.6.2. sn−2 Fatty Acid Composition Analysis
The sn−2 monoacylglycerols (sn−2 MAG) were prepared according to a published method [21]. A 30 mg lipid sample was mixed with a Tris-HCl buffer solution of sodium cholate and CaCl2, followed by the addition of pancreatic lipase to initiate enzymatic hydrolysis at 37 °C for 10 min. The reaction mixture was then extracted with 3 mL of diethyl ether, and after centrifugation at 4000× g for 10 min at 4 °C, the supernatant was collected. The products were separated using a silica gel 60 thin-layer chromatography (TLC) plate with a mobile phase of petroleum ether/diethyl ether/acetic acid (85:15:1, v/v/v). The target band was scraped off and subsequently eluted with petroleum ether. Finally, the sn−2 FA methyl esters were prepared following the same procedure used for total FA analysis and analyzed by GC.
Relative percentages of each FA at the sn−2 position were calculated using the formula (A/B × 3) × 100, where A and B represent the content (%) of the FA at the sn−2 position and in the total TAG molecule, respectively. The multiplication factor of 3 accounts for the fact that a TAG molecule contains three fatty acyl positions [22].
2.6.3. Analysis of Phospholipid Fatty Acid Composition
The PLs were isolated according to a published method [23]. The separation of PLs was carried out via silica TLC, employing a mobile phase of petroleum ether/diethyl ether/acetic acid (85:15:1, v/v/v). The corresponding silica segment was scraped off and extracted with chloroform/methanol (2:1, v/v) to obtain the PLs. Finally, the PLs were prepared following the same procedure used for total FA analysis and analyzed by GC.
2.7. Lipidomics Analysis
Lipid analysis was conducted using UPLC coupled to a Q Exactive Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Fisher Scientific, Bremen, Germany). Separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm; Waters, Milford, MA, USA) maintained at 40 °C. The mobile phase consisted of (A) acetonitrile/water (75:25, v/v) and (B) isopropanol/acetonitrile (8:2, v/v), both containing 0.1% formic acid and 10 mM ammonium formate. The elution gradient was as follows: 0 min, 15%B; 2 min, 30%B; 3 min, 48%B; 10 min, 70%B; 20 min, 99%B, 26 min, 15%B; 30 min, 15%B, delivered at 0.15 mL/min. The injection volume was 5 μL. Electrospray ionization (ESI) was employed with spray voltage set to 3000 V in positive ion mode and –2800 V in negative ion mode. Sheath gas, auxiliary gas, and sweep gas flow rates were 35, 15, and 1 arb, respectively. Both capillary temperature and vaporizer temperature were maintained at 350 °C, and the S-Lens RF Level was set to 50%. Mass spectra were acquired across an m/z range of 50–1300. Lipid identification was performed using high-resolution mass spectrometry data. Lipids were identified by matching their accurate mass (mass error < 5 ppm) and MS/MS spectra against a public lipid database (Lipid Search). The identification criteria required the presence of characteristic fragment ions and adduct forms specific to each lipid class.
2.8. Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were performed by one-way ANOVA followed by Tukey’s post hoc test, with a p < 0.05 considered statistically significant. Lipids satisfying all of the following criteria were defined as SDLs: variable importance in projection (VIP) > 1.0, fold change (FC) either < 0.33 or > 3.0, and p < 0.05. Multivariate statistical analyses, including principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), as well as visualization by volcano plots and heatmaps, were conducted using Metware Cloud (version 4.2), a free online data analysis platform. The OPLS-DA model was validated by 200-response permutation testing.
3. Results
3.1. Total Lipid and Phospholipid Content in Milk
The total lipid and PL contents in the milk of canine, feline, bovine and caprine are shown in Figure 1. Feline milk exhibited the highest total lipid content at 110.83 mg/mL, which was significantly greater than that of canine milk (81.52 mg/mL) and approximately three times higher than that of bovine (36.25 mg/mL) and caprine (40.27 mg/mL) milk. Regarding PL content, both canine (0.97 mg/mL) and feline (0.90 mg/mL) milk contained significantly higher levels, approximately threefold, compared to the values around 0.30 mg/mL found in bovine and caprine milk.
Figure 1.
Total lipid content (a) and phospholipid content (b) of milk from canine, feline, bovine, and caprine. Data are presented as Mean ± SEM (n = 4). Different letters indicate significant differences among groups based on one-way ANOVA followed by Tukey’s post hoc test (p < 0.05); groups sharing the same letter are not significantly different.
3.2. Fatty Acid Composition of Total Lipids and Phospholipids in Milk
The FA profiles vary considerably among milk sourced from different mammals. Table 1 breaks down the proportional concentrations of FAs found in canine, feline, bovine, and caprine milk fats, categorizing them into three main classes: saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA). The FA profiles of canine and feline milk were predominantly characterized by C16:0, C18:1n−9, and C18:2n−6. In contrast, bovine and caprine milk primarily contained myristic acid (C14:0), C16:0, stearic acid (C18:0), and C18:1n−9. These compositional differences were directly reflected in the total UFA content. The UFA content was markedly elevated in canine (71.66%) and feline (72.22%) milk relative to the levels in bovine (31.13%) and caprine (30.29%) milk. A particularly notable difference was observed in the PUFA composition. Feline milk registered the highest PUFA concentration, clearly outpacing canine milk and far exceeding found in bovine and caprine milk. This mainly results from elevated C18:2 levels in feline milk. Furthermore, regarding n−3 PUFAs, functional long-chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA, C20:5n−3) and docosahexaenoic acid (DHA, C22:6n−3) were detected in both canine and feline milk but were not found in bovine or caprine milk.
Table 1.
Fatty acid profiles of total lipids in canine, feline, bovine, and caprine milk (n = 4).
| Fatty Acid (%) | Total Lipids | |||
|---|---|---|---|---|
| Canine Milk | Feline Milk | Bovine Milk | Caprine Milk | |
| C10:0 | – | – | 2.15 ± 0.25 b | 4.63 ± 1.52 a |
| C12:0 | 0.13 ± 0.04 c | 0.10 ± 0.01 c | 3.61 ± 0.11 b | 5.12 ± 0.25 a |
| C14:0 | 2.41 ± 0.07 c | 1.09 ± 0.14 d | 12.93 ± 0.09 a | 12.49 ± 0.33 b |
| C14:1 | 0.15 ± 0.02 b | 0.06 ± 0.02 c | 0.99 ± 0.07 a | 0.20 ± 0.04 b |
| C16:0 | 22.89 ± 0.74 b | 21.44 ± 1.98 b | 38.18 ± 0.3 a | 36.88 ± 1.01 a |
| C16:1 | 6.04 ± 0.18 a | 4.39 ± 0.24 b | 2.02 ± 0.18 c | 2.05 ± 0.09 c |
| C17:0 | 0.31 ± 0.08 b | 0.24 ± 0.03 b | 0.61 ± 0.02 ab | 1.02 ± 0.51 a |
| C17:1 | 0.34 ± 0.04 a | 0.45 ± 0.15 a | 0.29 ± 0.03 a | 0.29 ± 0.04 a |
| C18:0 | 2.60 ± 0.39 d | 4.91 ± 0.39 c | 10.97 ± 0.05 a | 7.22 ± 0.20 b |
| C18:1 | 40.05 ± 0.75 a | 37.08 ± 0.87 b | 24.47 ± 0.28 c | 22.89 ± 0.75 d |
| C18:2n−6 | 18.6 ± 0.64 b | 24.90 ± 1.94 a | 2.95 ± 0.34 c | 4.43 ± 0.25 c |
| C18:3n−6 | 0.52 ± 0.09 a | 0.11 ± 0.03 b | – | – |
| C18:3n−3 | 1.52 ± 0.13 a | 1.60 ± 0.43 a | 0.19 ± 0.00 b | 0.13 ± 0.03 b |
| C20:0 | – | – | 0.42 ± 0.05 b | 0.94 ± 0.20 a |
| C20:1 | 0.75 ± 0.51 a | 0.29 ± 0.04 b | – | – |
| C20:2 | 0.40 ± 0.07 a | 0.22 ± 0.03 b | – | – |
| C20:3 | 0.36 ± 0.09 a | 0.18 ± 0.04 b | – | – |
| ARA(C20:4n−6) | 1.53 ± 0.27 a | 1.27 ± 0.40 a | 0.22 ± 0.01 b | 0.29 ± 0.01 b |
| C20:5n−3 | 0.69 ± 0.10 a | 0.91 ± 0.24 a | – | – |
| DHA(C22:6n−3) | 0.69 ± 0.12 a | 0.75 ± 0.01 a | – | – |
| ∑SFA | 28.34 ± 0.56 b | 27.78 ± 1.64 b | 68.87 ± 0.64 a | 68.30 ± 1.94 a |
| ∑MUFA | 47.34 ± 0.53 a | 42.27 ± 0.57 b | 27.76 ± 0.50 c | 25.43 ± 0.72 d |
| ∑PUFA | 24.32 ± 1.06 b | 29.95 ± 2.19 a | 3.36 ± 0.33 c | 4.86 ± 0.27 c |
| ∑n−3PUFA | 2.90 ± 0.32 a | 3.26 ± 0.65 a | 0.19 ± 0.00 b | 0.13 ± 0.03 b |
| ∑n−6PUFA | 20.65 ± 0.81 b | 26.28 ± 1.59 a | 3.17 ± 0.33 c | 4.73 ± 0.24 c |
| ∑UFA | 71.66 ± 0.56 a | 72.22 ± 1.64 a | 31.13 ± 0.64 b | 30.29 ± 0.69 b |
| ∑n−6PUFAs/∑n−3PUFA | 7.16 ± 0.53 c | 8.24 ± 1.31 c | 16.93 ± 2.00 b | 37.47 ± 2.45 a |
| ARA/DHA | 2.22 ± 0.17 a | 1.69 ± 0.51 a | – | – |
Note: Data were presented as Mean ± SEM (n = 4). Different superscript letters within the same row indicate significant differences among milk types based on one-way ANOVA followed by Tukey’s post hoc test (p < 0.05); groups sharing at least one common letter are not significantly different. “–” indicates not detected.
The FA composition of PLs in milk is summarized in Table 2. The PL fractions across all four milk types were predominantly composed of C16:0, C18:0, C18:1n−9, and C18:2n−6. However, the concentration of C18:0 in PLs notably exceeded its level in the total lipid composition. Consistent with the trend observed in total lipids, the proportion of PUFAs in PLs was significantly higher in canine (28.46%) and feline (29.00%) milk compared to bovine (9.72%) and caprine (10.58%) milk, with particularly notable differences in n−3 and n−6 series PUFAs. Furthermore, the proportion of n−3 PUFAs in PLs exceeded that in total lipids. Feline milk PLs exhibited the highest n−3 PUFA content (10.49%), which was primarily attributed to high levels of EPA and DHA. A similar enrichment trend was observed for arachidonic acid (C20:4n−6, ARA), which was most abundant in canine milk PLs and outpaced its levels in all other milk counterparts.
Table 2.
Fatty acid profiles of phospholipids in canine, feline, bovine, and caprine milk (n = 4).
| Fatty Acid (%) | Phospholipids | |||
|---|---|---|---|---|
| Canine Milk | Feline Milk | Bovine Milk | Caprine Milk | |
| C12:0 | 0.24 ± 0.15 b | 0.13 ± 0.01 b | 0.73 ± 0.05 a | 0.58 ± 0.11 a |
| C14:0 | 0.65 ± 0.14 d | 1.32 ± 0.05 c | 4.55 ± 0.04 a | 2.23 ± 0.10 b |
| C16:0 | 24.05 ± 0.55 d | 30.08 ± 0.10 c | 36.38 ± 0.26 b | 40.06 ± 0.07 a |
| C16:1 | 1.78 ± 0.16 a | 1.49 ± 0.04 b | 1.17 ± 0.11 c | 0.86 ± 0.11 d |
| C17:0 | 0.39 ± 0.05 c | 0.41 ± 0.03 bc | 0.69 ± 0.16 b | 1.10 ± 0.24 a |
| C17:1 | 0.90 ± 0.36 a | 0.49 ± 0.01 b | 0.49 ± 0.02 ab | 0.42 ± 0.13 b |
| C18:0 | 20.52 ± 1.75 c | 18.42 ± 0.17 d | 22.30 ± 0.13 b | 24.42 ± 0.09 a |
| C18:1 | 20.00 ± 1.25 b | 17.02 ± 0.19 c | 22.56 ± 0.24 a | 18.07 ± 0.16 c |
| C18:2n−6 | 15.05 ± 0.97 a | 11.29 ± 0.05 b | 6.07 ± 0.03 c | 6.78 ± 0.14 c |
| C18:3n−6 | – | 0.33 ± 0.01 a | – | – |
| C18:3n−3 | 0.67 ± 0.27 a | 0.37 ± 0.09 b | – | – |
| C20:0 | – | 1.12 ± 0.04 a | 0.55 ± 0.02 c | 0.85 ± 0.01 b |
| C20:1 | 3.01 ± 0.39 a | 0.53 ± 0.21 b | 0.85 ± 0.20 b | 0.82 ± 0.30 b |
| C20:3 | 0.28 ± 0.09 b | 0.60 ± 0.22 a | – | – |
| ARA(C20:4n−6) | 7.61 ± 0.90 a | 3.53 ± 0.02 b | 0.87 ± 0.05 c | 0.70 ± 0.03 c |
| C20:5n−3 | 0.96 ± 0.12 b | 5.08 ± 0.24 a | – | – |
| C22:2 | 1.13 ± 0.28 b | 2.76 ± 0.62 a | 2.78 ± 0.20 a | 3.10 ± 0.76 a |
| DHA(C22:6n−3) | 2.76 ± 0.21 b | 5.04 ± 0.41 a | – | – |
| ∑SFA | 45.85 ± 2.05 d | 51.47 ± 0.25 c | 65.20 ± 0.37 b | 69.25 ± 0.48 a |
| ∑MUFA | 25.69 ± 1.49 a | 19.53 ± 0.40 b | 25.08 ± 0.16 a | 20.17 ± 0.48 b |
| ∑PUFA | 28.46 ± 0.60 a | 29.00 ± 0.61 a | 9.72 ± 0.21 b | 10.58 ± 0.81 b |
| ∑n−3PUFA | 4.39 ± 0.55 b | 10.49 ± 0.21 a | – | – |
| ∑n−6PUFA | 22.67 ± 0.38 a | 15.15 ± 0.04 b | 6.94 ± 0.02 d | 7.48 ± 0.16 c |
| ∑UFA | 54.15 ± 2.05 a | 48.53 ± 0.25 b | 34.80 ± 0.37 c | 30.75 ± 0.48 d |
| ∑n−6PUFAs/∑n−3PUFA | 5.16 ± 0.42 a | 1.44 ± 0.18 b | – | – |
Note: Data were presented as Mean ± SEM (n = 4). Different superscript letters within the same row indicate significant differences among milk types based on one-way ANOVA followed by Tukey’s post hoc test (p < 0.05); groups sharing at least one common letter are not significantly different. “–” indicates not detected.
3.3. Lipidomic Profiles in Milk
In this study, lipidomic profiles of milk samples from canine, feline, bovine, and caprine species were obtained using both positive and negative ionization modes (Figure 2). In total, 2708 lipid species were identified and assigned to 17 distinct classes. TAGs were the most abundant in terms of the number of molecular species, comprising 1689 species and accounting for 62.37% of all identified lipid species. This was followed by phosphatidylethanolamine (PE, 258 species, 9.53%), ceramide (Cer, 146 species, 5.39%), phosphatidylcholine (PC, 109 species, 4.03%), and diacylglycerol (DG, 84 species, 3.10%). Other notable classes included phosphatidylserine (PS, 2.73%), sphingomyelin (SM, 2.55%), hexosylceramide (Hex1Cer, 2.14%), phosphatidylinositol (PI, 1.96%), cardiolipin (CL, 1.74%), and lysophosphatidylcholine (LPC, 1.00%). Additionally, minor lipid classes were detected (<1%), such as lysophosphatidylethanolamine (LPE, 0.81%), dimethylphosphatidylethanolamine (dMePE, 0.74%), phosphatidylglycerol (PG, 0.66%), dihexosylceramide (Hex2Cer, 0.63%), methylphosphatidylcholine (MePC, 0.33%), and sphingosine (SPH, 0.30%). Notably, both canine and feline milk individually contained over 100 more lipid molecular species than either bovine or caprine milk. Furthermore, TAGs represented the highest proportion in canine milk, constituting 70.49% of its total identified lipid species.
Figure 2.
The identified lipid numbers between positive ion modes (POS) and negative ion modes (NEG) (a). Composition of lipid classes in canine (b), feline (c), bovine (d), and caprine milk (e), expressed as the percentage of each lipid class relative to the total number of lipid species identified in the respective milk type. Abbreviations: TAG, triacylglycerol; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidyl-ethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol; CL, cardiolipin; MePC, methyl phosphatidylcholine; dMePE, dimethyl phosphatidylethanolamine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; SM, sphingomyelin; Cer, ceramide; Hex1Cer, hexosylceramide; Hex2Cer, dihexosylceramide; SPH, sphingosine.
3.4. Triacylglycerol Composition in Milk
The TAG species relative abundances as a percentage of total TAGs in canine, feline, bovine, and caprine milk fats are summarized in Table 3. This table includes only those TAG species with relative abundances exceeding 1% in either canine or feline milk. Specifically, 19 TAG species were identified at abundances greater than 1% in canine milk, with the top five most abundant being Bu-P-O, Co-P-Po, L-P-ARA, O-L-Ln, and Bu-P-L. In feline milk, 24 species exceeded the 1% threshold, and the five most predominant were Bu-P-L, Bu-Po-O, Po-Co-Po, O-L-Ln, and L-L-L. It is noteworthy that these dominant TAG species in canine and feline milk were present at trace levels or absent in bovine and caprine milk. Further analysis revealed that palmitoyl-esterified TAGs accounted for a substantially high proportion across all milk samples (Figure 3a). The highest level was observed in canine milk, reaching 41% of total TAGs, followed by bovine and feline milk, while caprine milk showed the lowest content.
Table 3.
TAG composition of milk from canine, feline, bovine, and caprine species (n = 4).
| TAGs (%) | Canine Milk | Feline Milk | Bovine Milk | Caprine Milk |
|---|---|---|---|---|
| Bu-P-O | 1.61 ± 0.19 a | 1.40 ± 0.08 a | 0.97 ± 0.11 b | – |
| Co-P-Po | 1.58 ± 0.22 a | 1.37 ± 0.10 a | – | – |
| L-P-ARA | 1.47 ± 0.55 a | 1.60 ± 0.18 a | 0.02 ± 0.01 b | 1.48 ± 0.09 a |
| O-L-Ln | 1.43 ± 0.61 a | 1.64 ± 0.18 a | 0.02 ± 0.01 b | 0.02 ± 0.03 b |
| Bu-P-L | 1.40 ± 0.21 a | 1.76 ± 0.14 a | 0.57 ± 0.10 b | – |
| Po-Co-Po | 1.38 ± 0.19 a | 1.71 ± 0.16 a | – | 0.19 ± 0.02 b |
| Bu-Po-O | 1.34 ± 0.23 a | 1.76 ± 0.13 a | 0.52 ± 0.09 b | – |
| O-P-Ln | 1.34 ± 0.35 a | 1.07 ± 0.39 a | 0.09 ± 0.01 b | – |
| L-L-L | 1.31 ± 0.85 a | 1.62 ± 0.18 a | – | 1.44 ± 0.25 a |
| P-P-ARA | 1.28 ± 0.34 a | 0.90 ± 0.25 a | 0.07 ± 0.02 c | 0.26 ± 0.04 b |
| O-L-L | 1.26 ± 0.21 a | 1.01 ± 0.19 a | 0.04 ± 0.00 b | – |
| O-P-ARA | 1.24 ± 0.21 a | 1.03 ± 0.19 a | 0.05 ± 0.01 b | – |
| Bu-O-O | 1.17 ± 0.14 a | 1.03 ± 0.06 a | 0.46 ± 0.06 b | – |
| Po-Co-O | 1.17 ± 0.14 a | 1.02 ± 0.07 a | 0.46 ± 0.07 b | – |
| L-P-Ln | 1.11 ± 0.11 a | 1.32 ± 0.42 a | 0.05 ± 0.02 b | – |
| Po-L-L | 1.10 ± 0.12 a | 1.27 ± 0.44 a | 0.03 ± 0.03 b | 0.03 ± 0.01 b |
| Po-P-ARA | 1.10 ± 0.11 b | 1.61 ± 0.30 a | – | – |
| L-P-L | 1.03 ± 0.29 a | 0.81 ± 0.39 a | 0.08 ± 0.01 b | – |
| O-Po-L | 1.00 ± 0.28 a | 0.82 ± 0.38 ab | 0.09 ± 0.02 c | 0.42 ± 0.10 b |
| Bu-O-L | 0.99 ± 0.14 b | 1.59 ± 0.12 a | 0.30 ± 0.06 c | 0.97 ± 0.09 b |
| L-L-Ln | 0.79 ± 0.06 b | 1.12 ± 0.21 a | – | 1.44 ± 0.09 a |
| L-P-EPA | 0.78 ± 0.05 a | 1.06 ± 0.32 a | – | 0.85 ± 0.17 a |
| O-P-L | 0.75 ± 0.40 a | 1.05 ± 0.27 a | 0.20 ± 0.04 b | 0.01 ± 0.02 c |
| Po-O-EPA | 0.60 ± 0.40 ab | 1.05 ± 0.31 a | – | 0.52 ± 0.03 b |
| Bu-L-L | 0.53 ± 0.07 b | 1.38 ± 0.12 a | 0.14 ± 0.03 c | 0.13 ± 0.01 c |
| Po-Co-L | – | 1.57 ± 0.10 a | 0.30 ± 0.06 c | 0.71 ± 0.07 b |
| Co-M-O | – | 1.37 ± 0.08 a | 0.97 ± 0.11 b | – |
Note: Bu, butyric acid; Co, caproic acid; M, myristic acid; P, palmitic acid; Po, palmitoleic acid; O, oleic acid; L, linoleic acid; Ln, linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid. Data were presented as Mean ± SEM (n = 4). Different superscript letters within the same row indicate significant differences among milk types based on one-way ANOVA followed by Tukey’s post hoc test (p < 0.05); groups sharing at least one common letter are not significantly different. “–” indicates not detected.
Figure 3.
TAGs containing palmitic acid in canine, feline, bovine, and caprine milk (a). The interpositional distribution of palmitic acid at the sn−2 position of TAGs among canine, feline, bovine, and caprine milk (b). Data are presented as Mean ± SEM (n = 4). Letters denote multiple-comparison groupings from one-way ANOVA followed by Tukey’s post hoc test. Groups sharing at least one common letter are not significantly different, whereas groups with different letters are significantly different (p < 0.05).
The sn−2 FA composition of milk samples was analyzed by GC, and the relative proportion of each FA at the sn−2 position was calculated (Table S1). Consistent with the total FA profile, canine and feline milks were predominantly composed of C16:0, C18:1n−9, and 18:2n−6, with their UFA content being significantly higher than that in bovine and caprine milks. Notably, the SFA proportion at the sn−2 position in canine and feline milks demonstrated a marked elevation over the level found in the total lipid fraction. Further calculation revealed that the sn−2 position in canine (57.78%) and feline (53.78%) milks was predominantly esterified by SFA, a characteristic not observed in bovine or caprine milk. Of particular interest, C16:0 predominated among the SFAs across all milk samples. As shown in Figure 3b, over 60% of palmitic acid was esterified at the sn−2 position of TAG molecules in canine and feline milks, whereas in bovine and caprine milks, only about 30% of palmitic acid was esterified at this position, indicating a strong sn−2 positional preference of palmitic acid in canine and feline milks.
3.5. Phospholipid Molecular Species in Milk
Table S2 presents the major PL molecular species with relative abundances exceeding 1% in canine and feline milk, respectively. The PC profile of canine and feline milk was enriched with PC (16:0_18:2), whereas bovine and caprine milk was characterized by a predominance of PC (16:0_18:1). The most abundant PE molecular species across all milk types was PE (36:2), specifically represented as PE (18:0_18:2) in canine, feline, and bovine milk, and as PE (18:1_18:1) in caprine milk. For PS, PS (18:0_18:2) was the dominant species in canine and feline milk, whereas PS (18:0_18:1) predominated in bovine and caprine milk. Regarding PI, the most abundant species in canine milk was PI (18:0_18:2), in feline milk it was PI (18:0_20:4), and in both bovine and caprine milk it was PI (18:0_18:1). In addition, SM with the structure SM(d18:1_16:0) was the most abundant SM species in all milk types, and most SM molecules contained the d18:1 sphingoid base. Notably, SM containing a 24:1 fatty acyl chain ranked as the second most abundant SM in canine and feline milk, with significantly higher relative levels than those in bovine and caprine milk. As for Cer, a higher number of Cer molecular species were identified in canine and feline milk compared to bovine and caprine milk, with Cer (d16:0_18:0) and Cer (d16:0_16:0) being the predominant species. Among lysophospholipids, LPC was most abundant as LPC (16:0) in canine, feline, and bovine milk, while in caprine milk it was LPC (18:1). For LPE, LPE (18:1) was the predominant species in all milk types.
3.6. Differential Analysis of Milk Lipids
To further investigate differential biomarkers among the four types of milk, this study integrated both univariate and multivariate statistical approaches to systematically screen for differential lipids. PCA was first employed to examine the overall distribution trends of the samples. The results demonstrated that PC1, PC2, and PC3 clearly separated canine, feline, bovine, and caprine milk (Figure 4a). Furthermore, OPLS-DA was applied for class discrimination. As shown in Figure 4b, the lipid compositions of the four milk types exhibited significant separation.
Figure 4.
The principal component analysis (PCA) score plot (a) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot (b) of canine, bovine, caprine, and feline milk; volcano plots comparing canine vs. bovine (c), canine vs. caprine (e), feline vs. bovine (g), feline vs. caprine (i), and canine vs. feline milk (k); along with heatmap analyses for each corresponding pairwise comparison (d,f,h,j,l), in which the top 40 lipids characterized by high abundance and significant differences were selected for display.
Differential lipid screening was performed for five comparison groups: canine–bovine, canine–caprine, feline–bovine, feline–caprine, and canine–feline. The threshold criteria were set as follows: FC > 3 or < 0.33, VIP > 1, and p < 0.05. The results are visualized in volcano plots (Figure 4c,e,g,i,k). Specifically: 712 differential lipids were identified in the canine–bovine comparison (360 up-regulated, 352 down-regulated); 722 differential lipids were found in the canine–caprine comparison (377 up-regulated, 345 down-regulated); 762 differential lipids were identified in the feline–bovine comparison (416 up-regulated, 346 down-regulated); 789 differential lipids were observed in the feline–caprine comparison (446 up-regulated, 343 down-regulated); 475 differential lipids were detected in the canine–feline comparison (198 up-regulated, 277 down-regulated). Hierarchical clustering analysis was further conducted to visually display the expression patterns of differential lipids among samples (Figure 4d,f,h,j,l). Differential lipids were ranked based on relative abundance, and the top 10 up-regulated and top 10 down-regulated neutral lipids and polar lipids, respectively, were selected, resulting in a total of 40 lipids for in-depth analysis. Notably, in both the canine–bovine and canine–caprine comparisons, TAG (18:1_18:2_18:3), TAG (16:0_18:2_20:4), and TAG (16:0_18:1_18:3) consistently exhibited high abundance and significant differences. The same trend was observed in the feline–bovine and feline–caprine comparisons. In the canine–feline comparison, lipids such as TAG (16:0_6:0_18:1) (FC = 6.91), TAG (4:0_18:0_18:1), TAG (14:0_18:2_18:2) (FC = 3.90), and TAG (16:1_6:0_14:0) (detected in one group but not the other) showed high abundance and significant differences.
4. Discussion
This study systematically characterized and compared the lipid profiles of canine, feline, bovine, and caprine milk, revealing marked interspecies differences in lipid content, FA composition and distribution, and lipid molecular species.
Total lipid content in canine and feline milk was significantly higher than that in bovine and caprine milk, consistent with previous reports [6,7]. Observed discrepancies among studies may be attributed to biological factors such as breed, lactation stage, and maternal diet [24,25,26]. In line with cross-species comparisons [6,7], canine and feline milk contained a significantly higher proportion of UFAs than bovine and caprine milk. Although higher UFA intake is associated with improved cardiovascular health in humans [27], it is important to recognize that dogs and cats possess distinct lipid metabolic requirements. Notably, canine and feline milk were markedly enriched in LC-PUFAs, particularly ARA and DHA, which were detected at trace levels or not detected in bovine and caprine milk. In human infant nutrition, ARA and DHA are widely supplemented in infant formula, and their ratio is recognized as critical for neural and cognitive development [28]. The ARA/DHA ratios in canine milk (2.22) and feline milk (1.69) both fall within the range recommended for human infants (approximately 1:1–2:1). Although DHA supplementation has been shown to support visual and cognitive development in puppies [29,30,31], direct evidence is still needed to determine whether the specific ARA/DHA ratios observed in canine and feline milk represent optimal ratios for neonatal development in these species.
This study further revealed important interspecies differences in TAG structure. Over 60% of palmitic acid was esterified at the sn−2 position of TAGs in both canine and feline milk, a positional distribution pattern closely resembling that of human milk [32]. In humans, sn−2 palmitate has been demonstrated to enhance calcium and fat absorption, support bone mineralization, and improve intestinal microbiota [33,34]; similar benefits could be hypothesized for puppies and kittens, but this needs validation in controlled feeding trials. Beyond positional distribution, the dominant TAG molecular species differed between canine and feline milk and were largely absent in bovine and caprine milk, suggesting species-specific mammary lipid synthesis.
Lipidomic analysis identified 2708 lipid molecules and clearly distinguished the lipid profiles of the four milk sources. Multivariate analysis revealed several TAG species significantly enriched in canine and feline milk, particularly TAG (18:1_18:2_18:3), TAG (16:0_18:2_20:4), and TAG (16:0_18:1_18:3), which may serve as potential biomarkers for species origin discrimination. The enrichment patterns of LC-PUFAs and specific FA combinations in these TAGs may reflect active metabolic pathways in the canine and feline mammary gland and hold relevance for neonatal development. Furthermore, direct comparison between canine and feline milk revealed distinct lipid profiles, including differential abundances of TAG (16:0_6:0_18:1) and TAG (14:0_18:2_18:2), further supporting the need for species-specific milk replacer formulation. Beyond nutritional applications, the lipidomic fingerprint established in this study may also serve as a technical tool for verifying milk source authenticity in commercial products, ensuring consistency between labeled and actual composition.
When translating these compositional targets into milk replacer formulations, practical constraints must be carefully addressed. Achieving specific TAG structural characteristics, such as higher sn−2 palmitate content and the inclusion of TAGs containing LC-PUFAs, may necessitate the use of structured lipids or customized blending strategies. The practical feasibility of such approaches depends on ingredient availability, economic considerations, and process compatibility. Given that PUFA-rich oils are prone to oxidative deterioration during thermal processing and storage, formulation and packaging strategies should emphasize oxidative stability. Furthermore, ingredient selection and labeling claims must comply with relevant regulatory standards for companion animal foods. Any formulation modifications guided by lipidomic data should be substantiated through product-specific stability testing.
Several limitations of this study should be acknowledged. The sample size was relatively small (n = 4 per species), and all samples were collected from specific breeds at a single lactation stage (3–4 weeks postpartum); thus, extrapolation of the findings requires caution. In addition, dogs and cats differ in digestive physiology and lipid metabolism, and the nutritional effects of features such as sn−2 palmitate and LC-PUFA enrichment in their milk remain to be tested directly in controlled neonatal feeding studies. Future work should expand sample diversity across breeds and lactation stages, combine compositional profiling with functional outcomes, and explore lipid-structuring approaches that can translate the observed differences into practical milk-replacer design.
5. Conclusions
This study conducted a systematic lipidomic comparison of milk from dogs, cats, cows, and goats. The results demonstrate that canine and feline milk exhibit substantially higher total lipid and PL contents, with a FA profile predominantly composed of UFAs and enriched in LC-PUFAs (such as ARA and DHA), which are present at trace levels or absent in bovine and caprine milk. Notably, over 60% of palmitic acid in canine and feline milk is esterified at the sn−2 position of TAG, a structural feature similar to that of human breast milk. Lipidomic analysis identified a total of 2708 lipid molecules and further screened several lipid species specifically enriched in canine and feline milk as potential biomarkers. These findings provide a scientific foundation for developing pet-specific nutritional formulations using bovine and caprine milk as base materials. It should be noted that this study is based on descriptive analyses of samples from specific breeds and lactation stages; therefore, the functional implications of the observed lipid differences and their practical efficacy in nutritional applications require further investigation through rigorously controlled animal experiments.
Acknowledgments
The authors would like to express sincere gratitude to Min Wen for providing the precious canine and feline milk samples essential for this study. We are also deeply grateful to Yuming Wang for securing the financial support through various funding programs, which made this research possible.
Abbreviations
The following abbreviations are used in this manuscript:
| TAG | Triacylglycerol |
| PL | Phospholipid |
| FA | Fatty Acid |
| SFA | Saturated Fatty Acid |
| MUFA | Monounsaturated Fatty Acid |
| PUFA | Polyunsaturated Fatty Acid |
| LC-PUFA | Long-Chain Polyunsaturated Fatty Acid |
| DHA | Docosahexaenoic Acid |
| ARA | Arachidonic Acid |
| EPA | Eicosapentaenoic Acid |
| OPO | 1,3-Dioleoyl-2-palmitoyl-glycerol |
| PE | Phosphatidylethanolamine |
| Cer | Ceramide |
| PC | Phosphatidylcholine |
| DG | Diacylglycerol |
| PS | Phosphatidylserine |
| SM | Sphingomyelin |
| Hex1Cer | Hexosylceramide |
| PI | Phosphatidylinositol |
| CL | Cardiolipin |
| LPC | Lysophosphatidylcholine |
| LPE | Lysophosphatidylethanolamine |
| dMePE | Dimethylphosphatidylethanolamine |
| PG | Phosphatidylglycerol |
| Hex2Cer | Dihexosylceramide |
| MePC | Methylphosphatidylcholine |
| SPH | Sphingosine |
| Bu | Butyric Acid |
| Co | Caproic Acid |
| M | Myristic Acid |
| P | Palmitic Acid |
| Po | Palmitoleic Acid |
| O | Oleic Acid |
| L | Linoleic Acid |
| Ln | Linolenic Acid |
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani16050710/s1, Table S1: Fatty acid composition at the sn−2 position of milk TAG from canine, feline, bovine, and caprine species; Table S2: Relative abundance of molecular species of sphingomyelin, glycerophospholipids, and lysoglycerophospholipids in milk from canine, feline, bovine, and caprine.
Author Contributions
Writing—Original Draft, Y.C.; Software, Y.C.; Data curation, Y.C.; Methodology, J.Y.; Formal Analysis, C.W.; Supervision, Y.W.; Funding Acquisition, Y.W.; Validation, H.Z.; Formal Analysis, H.Z.; Conceptualization, X.Z.; Resources, M.W.; Project Administration, M.W.; Visualization, M.W.; Writing—Review and Editing, T.Z.; Funding Acquisition, T.Z. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Ethical approval for the experiments was granted by the Experimental Animal Ethics Committee of the Ocean University of China (approval no. SPXY2025050841).
Informed Consent Statement
Written informed consent has been obtained from the owner of the animals.
Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material.
Conflicts of Interest
The co-author Min Wen is an employee of Gambol Pet Group Co., Ltd. The authors declare that there are no other conflicts of interest.
Funding Statement
This work was supported by National Key Research and Development Program of China (No. 2024YFD2401603), the National Natural Science Foundation of China (No. 22408357), the Taishan Scholars Program (No. tstp20240812), Leading the Charge with Open Competition Project of the State Key Laboratory of Marine Food Processing and Safety Control (No. 20250101), and Youth Innovation Team Program of Universities in Shandong Province (No. 2023KJ040).
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material.