The research proposal of this investigation was evaluated and authorized by the Animal Care and Use Committee of Shanxi Agriculture University, Taigu, China, prior to study commencement (IACUC Issue No. SXAU EAW-2021C.FU.00301413).

The experiment was undertaken from August 2021 to December 2021 at a dairy farm (Datong Sifang Hi-Tech Dairy Farm, Datong, China). A randomized block design experiment was carried out with 40 primiparous Holstein dairy cows (662 ± 13.9 kg BW, 71.3 ± 6.82 DIM, 36.1 ± 1.32 kg/d milk yield) at the beginning of the study. The feeding experiment was conducted for 120 d, comprising a 15-d covariate period followed by a 15-d adaptation period and subsequent 90-d sampling period. The diet (Table 1) was formulated according to NRC (2001) recommendations for a 680 kg cow producing 35 kg/d of milk containing 35 g/kg of milk fat and 35 g/kg crude protein. Cows were blocked by DIM and milk yield together and were randomly divided into 1 of 4 groups in a randomized block design. Four treatments were control (0 g/d of LA per cow), low-LA (LLA; 100 g/d of LA per cow), medium-LA (MLA; 200 g/d of LA per cow) and high-LA (HLA; 300 g/d of LA per cow). The amount of LA added was based on previous study (Faciola and Broderick, 2014) who found that 1.3% of LA addition to the basal diet has no influence on DMI. The LA additive (feed grade, contained 996 g/kg of LA [C12:0], 1.5 g/kg of decanoic acid [C10:0], 0.8 g/kg of decanoic acid [C14:0] and 1.7 g/kg of others; Wuxi Odio Technology Development) was blended into the basal diet (Table 1). The basal diet and LA additive was mixed as total mixed ration (TMR) once daily. Cows were housed in a naturally ventilated, two-row, head-to-head, free-stall barn equipped with Calan gates Feeding System for monitoring individual intake. Cows were milked three times daily (at 05:30, 13:30, and 20:30); fed the corresponding TMR ad libitum; and had free access to water.

The cows were weighed on 2 consecutive days at 16:00 on d 1 of the covariate period, and d 1 and 90 of the sampling period. The provided and refused TMR were determined daily for each dairy cow during the entire experiment to estimate the DMI. Samples of TMR were collected every 5 d of the covariate period and every 10 d during the sampling period, and stored at −20℃ for subsequent analyses. Milk production was measured daily for each cow during the entire experiment. Milk samples were collected every 5 d of the covariate period and every 10 d during the sampling period from each milking on 3 consecutive milkings within a day. Concurrently, samples were collected and stored at 4℃ with antimicrobial agent 2-bromo-2-nitropropane-1, 3-diol for subsequent analyses. At 06:30 and 18:30 during d 1 to 15 of the covariate period and d 70 to 87 of the sampling period, all cows were dosed with 5 g of chromic oxide powder placed in a gelatin capsule to serve as a digestion marker. Approximately 250 g fecal samples were collected from each cow's rectum at 07:00, 13:00, 19:00, and 01:00 during d 8 to 15 of the covariate period and d 78 to 87 of the sampling period, and stored at −20℃ for subsequent analyses. During d 88 to 89, samples of TMR, refusals, and feces of each cow were composited, dried at 55℃ for 72 h, and mashed to pass through a 1 mm screen with a cutter mill.

At 06:30, 09:30, 12:30, and 15:30 on d 5 of the covariate period and d 44 and 89 of the sampling period, the ruminal fluid was collected from each animal via an oral stomach tube. The initial ruminal fluid of approximately 150 mL was discarded to minimize saliva contamination, whereafter the next 200 mL was preserved. The ruminal pH of each cow was determined once using a portable pH meter (5011B; Shanghai Shuo optoelectronic Technology Co., Ltd., China). The ruminal fluid samples were filtered through four layers of medical gauze. Thereafter, 5 mL of the filtrate was blended with 1 mL of 250 g/L meta-phosphoric acid before being stored at −20℃ for VFA determination. Furthermore, 5 mL filtrate was blended with 1 mL of 20 g/L sulfuric acid before being stored at −20℃ for the analysis of ammoniacal nitrogen. For microbial DNA extraction and enzymatic activity analyses, 50 mL filtrate was placed in liquid nitrogen and subsequently stored at −80℃.

At 10:30 on d 15 of the covariate period and d 90 of the sampling period, blood samples from each dairy cow were collected into 10 mL evacuated tubes (Serum separation gel coagulant tube, Hunan Liuyang Medical Instrument Factory, Liuyang, China) via the coccygeal vessel. Blood samples were transported to the laboratory to separate the serum by centrifugation at 2000 × g and 4℃ for 12 min, whereafter the serum samples were stored at −20℃.

For each cow in groups supplemented with 0 and 200 g/d LA per cow, mammary tissue biopsies were carried out from 16:00 to 20:00 on d 15 of the covariate period and d 90 of the sampling period. About 1 g of secretory tissues in the mammary gland of each cow was collected via surgical biopsy as described by Farr et al. (1996) from the midpoint section of the rear quarter. Tissue biopsy samples for total RNA extraction were rapidly frozen in liquid nitrogen and kept in a low temperature refrigerator at −80℃ for total RNA extraction.

The contents of DM (method 934.01), nitrogen (method 976.05), ether extract (EE; method 973.18), and crude ash (method 942.05) in TMR, refusal, and feces samples were determined according to the method of AOAC (2000). The organic matter (OM) content was calculated as the differences between the DM and crude ash contents. The NDF content was determined as elaborated by Van Soest et al. (1991), using heat-stable alpha-amylase and sodium sulfite, and expressed including residual ash. The ADF content was analyzed based on the method described in AOAC (2000, method 973.18). Calcium (Ca) and phosphorus (P) were determined via the disodium ethylenediaminetetraacetate complexometric titration method and ammonium vanadate molybdate colorimetric method according to the methods of National Standards of the People's Republic of China GB/T 6436-2018 (China National Standard, 2018a) and GB/T 6437-2018 (China National Standard, 2018b), respectively. The fat, true protein, and lactose contents of the milk were analyzed using a Milko Scan FT-120 unit (Foss Electric) based on the method described in AOAC (2000, method 972.16). An aliquot of milk was centrifuged to obtain the milk fat cake. The milk fat was then extracted using the procedure of (Hara and Radin, 1978), and transmethylation of the esterified fatty acids was performed according to the method of Chouinard et al. (1999). Fatty acid methyl esters (FAMEs) were used for gas chromatographic analysis of total fatty acids. According to the method of Liu et al. (2018), fatty acid composition was determined using gas chromatography (GC) on a CP SIL 88, 100 m × 0.25 mm × 0.25 μm capillary column (Agilent J&W Advanced Capillary GC Columns, the Netherlands) in an Agilent 7890 A (Agilent Technologies, Santa Clara, CA, USA) with an auto sampler, flame ionization detector and split injection. The standards were Supelco 37 Component FAME Mix C4–C24 Unsatures (Catalog No. Sigma, 18919-1AMP, Sigma–Aldrich, USA), Supelco PUFA No. 1 (Marine Source, Catalog No. 47033, Supelco Chemical, USA), Methyl trans-11C18:1 (Sigma 46905, Sigma–Aldrich, USA), Methyl cis-9, trans-11 CLA (catalog no. Matreya1255, Cayman Chemical, USA), and Methyl cis-5,8,11,14,17-eicosapentaenoic acid (catalog no. Supelco 44864, Supelco Chemical, USA). The FAME were identified by comparisons with the retention times of the standards. The chromium content of the feces was measured using atomic absorption spectrophotometry (AAA320N; Shanghai Yidian Instrument Co., Ltd., China) based on the method of Williams et al. (1962). Ruminal VFA conctent were determined using gas chromatography (GC-7890; Agilent Technologies, Santa Clara, CA, USA). According to the method of Weatherburn (1967), ammoniacal nitrogen content was analyzed by a colorimetric spectrophotometer (UV759; Qingdao Juchuang Instrument Co., Ltd., China). The samples of ruminal fluid for the measurement of enzymatic activity were immediately taken to the laboratory. Activities of cellobiose, carboxymethyl cellulase [CMCase], α-amylase, xylanase, pectinase, and protease were determined as described by Agarwal et al. (2002). Biochemical kits (Nanjing Jiancheng Bioengineering Institute) of glucose (no. A154-2-1), total protein (A045-3-1), albumin (no. A028-1-1), blood urea nitrogen (BUN; no. C013-2-1), triglyceride (no. A110-2-1) and non-esterified fatty acids (NEFA; no. A042-2-1) were used to analyze serum content of glucose, total protein, albumin, urea nitrogen, triglyceride) and NEFA by using an automatic biochemical analyzer (BS-400, Nanjing Badeng Co., Ltd. China), The ELISA kits (Beijing Biolide Biotechnology Co., Ltd. China) of estradiol (E2; bovine, no. BL-E28820M), prolactin (bovine, no. BL-E28845M), and insulin-like growth factor 1 (IGF-1; bovine, no. BL-E21687M) were used to analyze E2, prolactin, and IGF-1 by using a Konelab auto-analyzer (Thermo Fisher Scientific, USA).

Exactly 1.5 mL of homogeneous ruminal fluid was used to extract microbial DNA using the repeated bead-beating plus column (RBB + C) as elaborated by (Yu and Morrison, 2004). The integrity and purity of the extracted DNA were evaluated via agarose gel electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Scientific, NanoDrop Technologies), respectively. The target microbial population comprised total bacteria, protozoa, anaerobic fungi, methanogens, R. flavefaciens, Ruminococcus albus, Butyrivibrio fibrisolvens, F. succinogenes, Ruminobacter amylophilus, and Prevotella ruminicola. The target microbial primer set sequences are listed in Table 2. For absolute quantification of the gene copy numbers, 10 sample-derived standards were prepared from the microbial DNA treatment pool using conventional PCR. The PCR products were purified, using the PureLink Quick Gel Extraction and PCR Purification Combo Kit (Thermo Fisher Scientific, USA), and quantified using a spectrophotometer. The copy number of each standard was calculated using the mass concentration and length of the PCR products (Yu et al., 2004). The target DNA was quantified using 10-fold serial dilutions from 10¹ to 10⁸ DNA copies. The real-time PCR amplification and detection was conducted in triplicate using a Chromo 4 system (Bio-Rad). The 20 μL reaction mixture contained 10 μL SYBR Premix Taq II (TaKaRa Bio), 2 μL DNA template, 0.8 μL forward primer (10 μmol/L), 0.8 μL reverse primer (10 μmol/L), 0.4 μL of ROX Reference Dye II (TaKaRa), and 6.0 μL dH₂O. The PCR cycling conditions were detailed as follows: 1 cycle at 50℃ for 2 min and 95℃ for 2 min for the original denaturation step, followed by 40 cycles at 95℃ for 15 s, anneal at annealing temperature of each target microbial DNA for 30 s, and extension at 60℃ for 1 min.

Six mammary tissue samples were selected randomly from the same block of two groups, from which total protein was extracted from 100 mg of ground bovine mammary tissue using radioimmunoprecipitation assay buffer (RIPA buffer) containing protease/phosphatase inhibitors (Roche Diagnostics, Shanghai, China). Protein content was analyzed using the Thermo Scientific Pierce BCA protein assay kit (23225; Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Beta-actin was used as the loading control. Equal quantities of protein (20 μg) were separated on a 12% SDS-PAGE, whereafter the separated proteins were transferred onto nitrocellulose membranes (1704271; Bio-Rad). The membranes were blocked with 6% (wt/vol) BSA in tris-buffered saline plus Tween (TBST) for 2 h at 25℃. Thereafter, the membranes were incubated overnight at 4℃ with the following primary antibodies diluted in TBST: mouse anti-cyclin A1 (1:2000; cat. no. NB100–2660; Novusbio Biologicals, USA), mouse anti-proliferating cell nuclear antigen (PCNA; 1:2000; cat. no. 2586; Cell Signaling Technology, USA), rabbit anti-Akt (1:2000; cat. no. 9272; Cell Signaling Technology, USA), rabbit anti-phosphorylated-Akt_Ser473 (1:2000; cat. no. bs-0876R; Bios Antibodies, USA), rabbit anti-mTOR (1:2000; cat. no. bs-1992R; Bios Antibodies, USA), rabbit anti-phosphorylated-mTOR_Ser2448 (1:2000; cat. no. bs-3495R; Bios Antibodies, USA), rabbit anti- G-protein-coupled receptor 84 (GPR84; 1:2000; cat. no. bs-13507R; Bios Antibodies, USA), rabbit anti-peroxisome proliferator-activated receptor gamma (PPARγ, 1:2000; cat. no. bs-4590R; Bios Antibodies, USA), rabbit anti-sterol regulatory element-binding protein 1 (SREBP1; 1:2000; cat. no. NB100–2215; Novus Biologicals, USA), rabbit anti-phosphorylated-acetyl-coenzyme A carboxylase-α (ACACA; 1:2000; cat. no. bs-12954R; Bios Antibodies, USA), rabbit anti-ACACA (1:2000; cat. no. bs-11912R; Bios Antibodies, USA), rabbit anti-fatty acid synthase (FASN, 1:2000; cat. no. bs-60347R; Bios Antibodies, USA), rabbit anti-stearoyl-CoA desaturase 1 (SCD1; 1:2000; cat. no. bs-3787R; Bios Antibodies, USA), and rabbit anti-β-actin (1:10,000; cat. no. 4970; Cell Signaling Technology, USA). In order to remove excess antibodies, membranes were washed five times with 1 × TBST for 5 min. Thereafter, the membranes were incubated at 25℃ for 2 h with either of the following secondary antibodies diluted in TBST: goat anti-rabbit (1:5000; cat. no. E-AB-1003; Elabscience, USA) or goat anti-mouse (1:5000; cat. no. RS0001; Immunoway, USA). SuperSignal West Pico Chemiluminescence Substrate (Thermo Fisher Scientific, USA) was used to visualize the western blots prior to being semi-quantified using the ImageJ software (version 1.4.3.67).

Dietary net energy for lactation was estimated by multiplying the NE_L-3X density of the feed ingredient by its dietary content (NASEM, 2021). Energy-corrected milk (ECM) and fat-corrected milk (FCM) were estimated according to NRC (2001), where ECM = 0.327 × milk (kg/d) + 12.95 × fat (kg/d) + 7.65 × protein (kg/d), 4% FCM = 0.4 × milk (kg/d) + 15 × fat (kg/d). Feed efficiency was estimated for each animal as milk yield (actual milk and ECM yield) divided by dietary DM intake. Nutrient digestibility (%) was calculated by the following formula: (1 - bc/ad) × 100, where a was nutrient concentration in feed; b was nutrient concentration in feces; c was chromium concentration in feed; d was chromium concentration in feces (Salehi et al., 2023).

Data were analyzed as a randomized complete-block design via the mixed procedure of SAS (PROC MIXED; SAS, 2002). Data for DMI, milk production, feed efficiency and milk fatty acid composition were analyzed using the following statistic model:where Y_iklm was the dependent variable; μ was the overall mean; β was regression coefficient; V_k was covariate measurement; B_i was the random effect of block i; T_j was the fixed effect of time j; C_k(i) was the random effect of cow k within block i; L_l was the fixed effect of LA treatment l; TL_jl was interaction between time and LA treatment; E_ijkl was residual error.

Data for rumen fermentation, microbial enzyme activity and microbiota were analyzed using the following statistic model which there were repeated measurements over time (pH, VFA, enzymatic activity and microbiota):where, Y_iklm was the dependant variable; μ was the overall mean; β was regression coefficient; V_k was covariate measurement; B_i was the random effect of block i; D_j was the fixed effect of day j; C_k(i) was the random effect of cow k within block i; L_l was the fixed effect of LA treatment l; DL_jl was interaction between day and LA treatment; E_ijkl was whole plot error; T_m was effect of time m; LT_ml was interaction between LA treatment and time; S_ijklm was subplot error. Original data was tested in SAS for homogeneity of variance and normality; moreover, rumen microbiota residuals were tested for normality. Log-transformed microbiota data only marginally improved normality; therefore, analysis was done using original data. The covariance structure for variables was first-order autoregressive. Mean separations using probability of difference tests (PDIFF in SAS) were conducted only for influences that were statistically significant (P < 0.05). Data for nutrient digestibility and blood parameters were analysed with the same model as suggested above, but time and the interaction between time and treatment were omitted. The CONTRAST statement of SAS was used to compute linear and quadratic orthogonal contrasts based on the LA application rates.

SigmaPlot version 12.5 statistical analysis package (Systat Software, Inc., San Jose, CA, USA) was used to analyze the western blotting results. Student's t-test (t-test) was used to analyze statistical differences between treatments. Effects of the factors were considered significant at P < 0.05 unless other trends were declared at P < 0.10.

The DMI decreased linearly (P = 0.042) with increased LA addition (Table 3). Conversely, the yields of actual milk (P = 0.036), 4% FCM (P = 0.037), and ECM (P = 0.042) increased quadratically with an increase in LA addition (Table 3). Milk fat content (P = 0.014) and production (P = 0.038) increased linearly with increased LA supplementation, whereas milk true protein content (P = 0.014) and production (P = 0.013) increased quadratically following LA supplementation. However, the production and content of milk lactose did not change with increased LA addition. Feed efficiency, described as milk yield to dietary DM intake ratio (P = 0.032) or ECM yield to dietary DM intake ratio (P = 0.019), also increased linearly with increased LA dose.

Supplementation of LA had an effect on de novo fatty acids (DN FA; Σ FA < 16C; P = 0.038) (Table 4). Most of the increase in DN FA was depend on an elevation in the proportion of C12:0 in milk (P = 0.018). However, supplementation of LA decreased (P = 0.045) the proportions of mixed sourced fatty acids (MSFA; Σ FA = 16C) in milk due to the linearly decreased proportion of C16:0 in milk (P = 0.046). Although supplementation of LA increased the proportions of C18:1 (P = 0.038) and C20:1 (P = 0.046) in milk, there were no difference in the proportions preformed fatty acids (PFA; Σ FA > 18C). Proportions of odd- and branched-chained fatty acids (OBCFA) was also not impacted by LA addition.

The digestibility of dietary DM, OM, NDF and ADF, increased linearly and quadratically (P < 0.05), but that of CP (P = 0.018) and EE (P = 0.024) increased linearly with increasing LA dose (Table 5). Although ruminal pH did not decrease, ruminal total VFA content increased quadratically (P = 0.034) with increased LA dose. The molar proportion of acetate was unaffected by LA addition, whereas that of propionate increased linearly (P = 0.021) with increased LA addition. Moreover, the acetate to propionate ratio decreased linearly (P = 0.018) with an increase in LA addition. Conversely, the molar percentages of butyrate, valerate, isobutyrate and isovalerate were unaffected by LA addition. Rumen ammonia N content decreased linearly (P = 0.024) with increased LA addition.

Although the enzyme activities of CMCase and cellobiase were unaffected by LA addition, the activity of xylanase increased linearly (P = 0.012) with increased LA dose (Table 6). The activity of pectinase (P = 0.022) and α-amylase (P = 0.008) increased linearly with an increase in LA dosage. Populations of total anaerobic fungi (P = 0.043) and bacteria (P = 0.044) increased linearly with an increase in LA dosage. Nevertheless, populations of total protozoa (P = 0.019) and methanogens (P = 0.017) decreased linearly with an increase in LA dosage. Populations of R. flavefaciens (P = 0.024), R. albus (P = 0.015), and R. amylophilus (P = 0.047) increased quadratically with an increase in LA dosage. However, populations of F. succinogenes, B. fibrisolvens, and P. ruminicola were unaffected by LA addition.

The blood levels of glucose (P = 0.030), triglyceride (P = 0.014), E2 (P = 0.032), prolactin (P = 0.024), and IGF-1 (P = 0.018) increased linearly with an increase in LA addition (Table 7). Furthermore, blood albumin (P = 0.016) and total protein (P = 0.013) concentrations increased quadratically with increased LA dose. Conversely, blood UN content decreased quadratically (P = 0.013) with increased LA dose. Furthermore, blood NEFA concentration decreased linearly (P = 0.013) with increased LA addition.

In regard to fatty acid synthesis, the protein levels of PPARγ (P < 0.01), SREBF1 (P < 0.01), FASN (P < 0.01), SCD1 (P < 0.05) and the phosphorylation ratio of ACACA were uniformly increased upon the addition of 200 g/d LA compared with the control (Fig. 1A and B).

The protein levels of cyclin A1 (P < 0.01) and PCNA (P < 0.05) were uniformly increased upon the addition of 200 g/d LA compared with those in the control (Fig. 2A and B). As a receptor for LA, the GPR84 regulates cell proliferation and apoptosis via the Akt/mTOR signalling pathway. The protein levels of GPR84 was increased (P < 0.01) following 200 g/d LA addition. The phosphorylation ratios of Akt and mTOR were also increased (P < 0.01) following 200 g/d LA addition (Fig. 3A and B).

The linear reduction in the DMI of dairy cows is in consonance with the results obtained by Dohme et al. (2004), who found that LA (C12:0) addition reduces DMI to a greater extent than the C14:0 and C18:0 addition. In keeping with the current findings, Külling et al. (2002) also found a decrease in DMI with LA addition. Moreover, 1.3% of LA addition to the basal diet has no influence on DMI (Faciola and Broderick, 2014); however, larger doses of LA in the TMR (480 and 720 g/d) drastically reduce DMI (Faciola and Broderick, 2013). The decreased DMI upon LA supplementation may be ascribed either to the poor palatability of LA (Külling et al., 2002) or impaired rumen degradation of fibre (Dohme et al., 2004) and resultant increased ruminal retention time of feed (Klop et al., 2017a). Previous studies reported that yields of milk and milk components is either unimpacted (Faciola et al., 2013; Hristov et al., 2009) or decreased (Hristov et al., 2011; Faciola and Broderick, 2014; Klop et al., 2017b) following LA supplementation. Conversely, we found that LA addition quadratically increased milk production of actual, 4% FCM, and ECM, quadratically increased production and content of milk protein, and linearly increased production and content of milk fat. This is mainly ascribed to the chosen LA dose, since Faciola and Broderick (2014) used an LA dose of 1.3% of dietary DM, whereas that in our study was 0.46% (LLA), 0.94% (MLA), and 1.45% (HLA) of dietary DM. Furthermore, the quadratic respond to LA addition with no further elevation of milk production indicate that increasing LA dose from 0.94% to 1.45% of dietary DM was not conducive to improving lactation performance. The quadratic response in milk production following LA supplementation might be due to the linear and quadratic changes in the digestibility of DM, OM, NDF and ADF, further research is needed to verify this explanation.

As expected, elevating the supply of C12:0 in the cow diet elevates the proportion of C12:0 which is inflating the proportions of DN FA. Similarly, Dohme et al. (2004) found a greater proportion of C12:0 in milk fat of cows following LA addition compared to C14:0 and C18:0. The decreased proportions of MSFA was mainly resulted from the decreased proportion of C16:0 in milk fat and was due to the decreased DMI. Other previous studies also observed the increased proportion of C12:0 in milk fat and the decreased proportion of C16:0 upon LA addition (Hristov et al., 2011; Klop et al., 2017a,b). The PFA are mostly coming from the diet and from the biohydrogenation of dietary FA. In the current study, the increased proportions of C18:1 and C20:1 in milk fat of cows following LA addition was due to an increase in the apparent digestibility of ether extract and an increase in stearoyl-CoA desaturase. Similarly, Klop et al. (2017a) found that proportions of several C18:1 fatty acids in milk fat of cows were increased with 65 g/kg DM of LA. The unchanged proportions of PFA, OBCFA, SFA, monounsaturated fatty acid (MUFA) or polyunsaturated fatty acids (PUFA) upon LA addition was in keeping with Klop et al. (2017a) and might be due to the comprehensive effect of the decreased DMI and increased nutrient digestibility following LA addition.

Previous researches demonstrated digestibility of dietary DM, OM, and CP was not impacted, and both the total-tract and ruminal apparent digestibility of dietary NDF and ADF was reduced upon LA supplementation (Faciola et al., 2013; Faciola and Broderick, 2013, 2014) and after coconut oil feeding (Lee et al., 2011) in dairy cows. However, in the current study the increased nutrient digestibility following LA addition was possibly depend on the increased ruminal bacterial populations and enzymatic activity with an increasing LA dosage (Liu et al., 2018), and resulted in the increased ruminal VFA concentrations, supporting the quadratically improved milk production. Furthermore, the quadratic effect of LA on dietary DM, OM, NDF and ADF digestibility might be depend on the quadratic response of populations of R. albus, R. flavefaciens and Rb. amylophilus to LA addition.

We demonstrated that ruminal pH was unaffected by an increase in LA addition, which concurs with previous observations (Faciola and Broderick, 2013, 2014). Similarly, Kim et al. (2018) demonstrated that LA addition did not impact ruminal pH in Hanwoo steers. Possible explanation for an increase in total VFA and a decrease in ammonia with no change in pH in the current study is an increased absorption of VFA (Suárez et al., 2006) or a net increase of VFA flus (Rémond et al., 2003). Since the VFA flus and production were not measured in this study, it is not possible to tell whether an increase in VFA flow is equivalent to absorption, which was also a limitation of this study. The increased total VFA content was in line with the increased dietary DM digestibility and suggested that ruminal bacterial populations and enzymatic activity were increased following LA addition. Moreover, the linearly decreased ratio of acetate to propionate was depend on the unaltered acetate and linearly increased propionate molar percentages. This implied that the ruminal fermentation pattern tended towards propionate formation under increased LA addition conditions. Correspondingly, Kim et al. (2018) demonstrated that total VFA and propionate content in LA-supplemented Hanwoo steers were greater than those of the control group 6 h after feeding. For glucose and lactose to be synthesized, propionate is an essential substrate (Wang et al., 2009a; Li et al., 2016); therefore, it is possible that LA supplementation enhanced milk production via this mechanism. Nevertheless, Faciola and Broderick (2014) demonstrated that 1.3% LA, based on the dietary DM of dairy cows, caused a small reduction in total VFA concentrations, although the ratio of acetate to propionate was decreased. The linearly decreased rumen ammonia nitrogen content is possibly ascribed to the increased bacterial protein synthesis with LA addition (Cummins and Papas, 1985).

In cows, dietary fiber is degraded to acetate by the ruminal bacteria, protozoa, and fungi that secrete cellulolytic enzymes (Orpin, 1984). Feed lignocellulosic tissues can be degraded by ruminal fungi, and approximately 30% of fiber digestion and 10% of VFA production could be due to ruminal protozoa (Wang and McAllister, 2002). Therefore, the linear increase in ruminal xylanase activity with an increasing LA dosage was a consequence of the increased populations of total anaerobic fungi, total bacteria, R. flavefaciens, and R. albus. Moreover, this result supported the increased rumen total VFA and increased digestibility of dietary NDF and ADF.

The increased populations of total anaerobic fungi, total bacteria, R. flavefaciens, and R. albus might be mainly due to the supply of energy from the degradable LA, since the effective degradability of rumen by-pass fat was about 86% (Soren et al., 2022). The linear reduction in populations of total methanogens and protozoa suggested that LA addition suppressed ruminal CH₄ production (Soliva et al., 2003). Correspondingly, Kim et al. (2018) observed that LA supplementation decreased methanogen and F. succinogenes populations and significantly increased R. flavefaciens populations. The LA elicits potent antiprotozoal effects (Lee et al., 2011; Faciola and Broderick, 2013) and 160 g/d of LA provided via ruminal cannula reduces ruminal protozoa proportion by 90% within 2 d of treatment (Faciola et al., 2013). The reduction of protozoa population resulted in the decreased protozoa's ability to phagocytose bacteria, thus changing the structure of microflora (Hristov et al., 2012), increasing the populations of total bacteria, total anaerobic fungi, and promoting ruminal fermentation (Wang et al., 2009b). The linear increase in the activity of α-amylase and pectinase was in agreement with the observed elevation in the R. amylophilus population, indicating that LA addition promoted ruminal starch degradation. These results further confirm the increased molar percentage of propionate with an increasing LA dosage. The unaltered ruminal protease activity was mainly associated with the unchanged P. ruminicola population.

The propionate produced by ruminal fermentation is transported to the liver and subsequently converted to glucose (Chan and Freedland, 1972). Therefore, the linear increase in blood glucose following LA addition, which is in keeping with the results of previous researches (Li et al., 2016; Wang et al., 2009a), may be ascribed to an increase in ruminal propionate output (Foley et al., 2009). The linear reduce in NEFA concentration and the linear increase in triglyceride concentration following LA supplementation indicated that body fat mobilization was suppressed and fatty acid synthesis was promoted (Coleman et al., 2021). The increase in glucose and the decrease in NEFA following LA supplementation might suggest an increase in insulin resistance, although some literature in monogastric animals appears to support a reverse effect (Tham et al., 2020), while others indicate an increase in insulin resistance in the adipose tissue (Saraswathi et al., 2020) and liver (Kamoshita et al., 2022). Total protein, albumin, and BUN concentrations are indicators of the protein utilization efficiency (Nousiainen et al., 2004). Although we observed the increased albumin and total protein levels and decreased BUN concentrations following LA supplementation, the ability of LA to improve protein utilization needed to be further studied. It is well-known that IGF-1, prolactin, and E2 are vital for mammary duct development (Rosen, 2012). Moreover, ovary-derived IGF-1, prolactin, and E2 stimulate epithelial cell proliferation (Mallepell et al., 2006). Additionally, the PI3K/Akt signaling pathway was activated by IGF-1 through its receptor and promotes the proliferation of epithelial cells (Zhou et al., 2017). Therefore, our results showing LA addition-induced increased serum IGF-1, prolactin, and E2 concentration can provide some support for a possible mechanism of the effect of LA on mammary epithelium. However, further research on the expression of gene and protein related to the above hormone and its receptor are needed to confirm the effect of LA on mammary epithelium.

Based on the quadratic increase of LA in promoting milk yield and ruminal total volatile fatty acid concentration of dairy cows, and the better effect of the MLA group was selected to compare the difference of proteins implicated in fatty acid synthesis and cell proliferation with the control group.

Both PPARγ and SREBP1 regulate the expression of ACACA, FASN, SCD, and fatty acid-binding protein 3 (FABP3) in the mammary gland (Ma and Corl, 2012). Moreover, both FASN and ACACA are involved in fatty acids synthesis from acetate and β-hydroxybutyric acid in the mammary gland (Bionaz and Loor, 2008) and are positively correlated with the secretion of C_4-16 fatty acids (Bernard et al., 2008). The SCD protein catalyzes MUFA synthesis from SFA (Lehner and Kuksis, 1996; Zhang et al., 2023). The increased protein levels of FASN, ACACA, PPARγ and SREBP1 following LA supplementation indicated that the proteins implicated in the synthesis of de novo FA and MCFA were upregulated, supporting the elevation in milk fat yield. Similarly, Busato and Bionaz (2021) demonstrated that PPARγ activated by LA in immortalized cell culture model of bovine liver.

Stimulating cell proliferation in the mammary gland should be the core control point that promotes lactation persistence in dairy cows. The Cyclin A is regarded as indispensable for initiating and completing DNA replication in the S phase (Lim and Kaldis, 2013). Additionally, PCNA is an essential component of both DNA replication and repair (Park et al., 2016). Moreover, previous researches have certificated the involvement of Cyclin D3 and PCNA in modulating mammary epithelial cell proliferation (Zhang et al., 2018). We demonstrated that LA addition increased the protein expressions of cyclin A1 and PCNA, implying that LA stimulates the proliferation of mammary epithelial cells. Traditionally, most of mammary development and cell growth and differentiation occurs before parturition and then net growth during lactation is expected to be minimal. In view of the results of the current experiment, it could provide a way to promote the research of mammary gland development of perinatal cows in the future.

Previous researches have implicated the Akt signaling pathway in modulation of the proliferation and apoptosis of various cells (Huang et al., 2021; Meng et al., 2017). Moreover, the mTOR signaling pathway is concerned with modulating the proliferation of various cells (Li et al., 2017). Existing research has shown that GPR84, which is strongly activated by LA (Miyamoto et al., 2016; Wang et al., 2006), regulates immune responses (Alvarez-Curto and Milligan, 2016) and MCFA taste transduction (Liu, 2016). Furthermore, LA addition increases GPR84 and Cyclin D1 expression and activates the PI3K/Akt signaling pathway of the mammary glands of pubertal (Meng et al., 2017) and lactating (Yang et al., 2020) mice. The addition of LA increased GPR84 expression and subsequently stimulated Akt and mTOR phosphorylation via GPR84 activation. The results suggest that activating the Akt-mTOR signaling pathway might stimulate proliferative markers to be expressed. Considering that the net growth of mammary gland during lactation is expected to be minimal, the activation of AKT and mTOR with LA addition would be likely more in metabolic regulation, especially the regulation of milk synthesis.