Small ruminants play a crucial role in global agriculture and natural resource management (NRC, 2007). Goats and sheep, as ruminants, play a critical role in the economic development and food security of various regions worldwide, particularly in tropical semi-arid zones, where goats are especially significant (Hernández-Castellano et al., Reference Hernández-Castellano, Nally, Lindahl, Wanapat, Alhidary, Fangueiro, Grace, Ratto, Bambou and Almeida2019). By 2030, the global population is expected to increase significantly, particularly in tropical countries. Given the projections for human population growth, particularly in tropical regions, it is essential that high-quality animal products, including meat and milk, be available to feed this population in the near future. Sheep and goats, therefore, play a crucial role in ensuring food security, alleviating poverty, and ultimately eradicating hunger. Small ruminants are not only associated with subsistence farming systems for dairy production, but with production in all types of systems. The improvement of knowledge about ruminant nutrition and the increasing adoption of technologies by farmers reveal new knowledge gaps that need to be filled. In this context, the use of feed additives that alter ruminal fermentation in dairy goat diets is an example of such technologies. Nutritional strategies have a great impact on animals’ growth (Teixeira et al., Reference Teixeira, Fernandes, Pereira Filho, Canesin, Gomes and Resende2017) and milk production (Chilliard et al., Reference Chilliard, Glasser, Ferlay, Bernard, Rouel and Doreau2007).
The incorporation of functional additives into ruminant diets and their subsequent effects on the lipid composition of milk have garnered significant scientific interest. Among these natural bioactive compounds are derivatives of the cashew tree (Anacardium occidentale L.), a tropical species belonging to the Anacardiaceae family. The cashew tree (Anacardium occidentale L.) is native to north-eastern Brazil and has since been introduced and extensively cultivated in various tropical and subtropical regions, including India, Mozambique, Tanzania, Kenya, Vietnam, Indonesia and Thailand. Its primary economic value lies in the production of cashew nuts. During nut processing, considerable quantities of cashew nut shell liquid (CNSL) are generated as a by-product. CNSL extraction is performed on an industrial scale through multiple techniques, including cold pressing, solvent extraction (Kumar et al., Reference Kumar, Paramashivappa, Vithayathil, Rao and Rao2002), thermomechanical processing and supercritical carbon dioxide (CO₂) extraction (Patel et al., Reference Patel, Bandyopadhyay and Ganesh2006).
CNSL is a viscous, oily, caustic, dark-brown substance that can be extracted from the cashew nut (Chaves et al., Reference Chaves, Cito, Lopes, Costa, Oliveira, Costa and Brito Junior2010), which accounts for 25% of the total weight. According to Trevisan et al. (Reference Trevisan, Pfundstein, Haubner, Wurtele, Spiegelhalder, Bartsch and Owen2006), CNSL is one of the richest sources of isoprenoid phenolic lipids such as anacardic acid (derived from salicylic acid), cardols and methyl-cardols (derived from resorcinol) and cardanols (a monophenol). Natural CNSL extracted by solvents contains anacardic acid (60–65%), cardol (15–20%), cardanol (10%) and traces of 2-methylcardol (Kumar et al., Reference Kumar, Paramashivappa, Vithayathil, Rao and Rao2002). In this sense, CNSL has aroused great interest in research studies in chemical and biological fields due to its high concentration of anacardic acid.
The CNSL is considered a by-product of cashew agribusiness, with very low added value. This liquid is one of the richest sources of naturally occurring non-isoprenoid phenolic lipids. The CNSL has been characterized as a bioactive compound with functional attributes and pronounced antioxidant activity, demonstrating potential when incorporated into ruminant feeding systems. Due to its renewable, biodegradable and sustainable nature, CNSL also represents a cost-efficient agro-industrial by-product, particularly within the Brazilian agribusiness context (Andrade et al., Reference Andrade, Araujo, Citó, Silva, Saffi, Richter and Ferraz2011; Kang et al., Reference Kang, Suzuki, Suzuki, Koike, Nagashima and Kobayashi2018; Michailoff et al., Reference Michailoff, Silveira, Maeda, Sordi, Francisco and Farenzena2020). According to Osmari et al. (Reference Osmari, Branco, Diaz, Matos, Goes and Teodoro2019), the dietary inclusion of technical CNSL, in combination with non-protein nitrogen sources, does not compromise nutrient digestibility or modulate ruminal fermentation patterns. In a complementary study, Branco et al. (Reference Branco, Giallongo, Frederick, Weeks, Oh and Hristov2015) administered 3% tCNSL in the diets of lactating dairy cows and observed no significant effects on nutrient digestibility or enteric methane emissions; the supplementation resulted in a 17% reduction in the concentration of stearic acid (C18:0) within the milk FA profile, without inducing detectable changes in methane production. However, there are few studies with natural CNSL on the lipid profiles of goat milk.
The hypothesis of this study was that inclusion CNSL would improve the milk FA profile and would not negatively affect feed intake or milk production in lactating goats. This study aimed to evaluate the effects of levels of CNSL in goats’ diets on animal performance, microbial modulation and milk quality.
Materials and methods
Site and ethics statement
The animal experiment was conducted at the Department of Animal Science of the Federal University of Ceara, in Fortaleza, Ceara State, Brazil (30°43′02″S, 33°32′35″W). During the experimental trial, the mean daily minimum and maximum air temperature and relative humidity were 24.6 °C ± 0.82 and 31.2 °C ± 1.32 and 71.1% ± 7.58 and 89.1% ± 4.27, respectively. Animal care and management were followed according to the guidelines and requirements of the Ethics Committee on Animal Research of the Federal University of Ceara, Fortaleza, Brazil (Protocol number 1,898,051,018).
Extraction of natural cashew nut shell liquid
To obtain the CNSL, the cashew shells were initially ground in a grinder (GTI-2000 LDF) and stored in glass containers. Subsequently, the cashew shells were immersed in 99.5°GL ethanol (Trevisan et al., Reference Trevisan, Pfundstein, Haubner, Wurtele, Spiegelhalder, Bartsch and Owen2006) in a 1:1 ratio for seven days at room temperature. After this period, the material was filtered through a 0.5 mm non-woven fabric, obtaining the cashew nut liquid (CNSL) plus 99.5°GL ethanol. The CNSL and ethanol were then evaporated in a rotary evaporator (Evaporador Rotativo TE–210®, Tecnal, Piracicaba, Brazil) at 50 °C, 60 rpm, and reduced pressure to recover the solvent (ethanol) and obtain pure CNSL. The CNSL containing 84.4% acid anacardic, 23.2% cardanol and 15.6% cardol.
The CNSL presented FA profile in 100 g total FA was 0.16 mg C6:0, 0.50 mg C8:0, 0.25 mg C10:0, 1.30 mg C12:0, 4.06 mg C14:0, 129.53 mg C16:0, 20.63 mg C16:1, 35.79 mg C18:0, 175.22 mg C18:1-c9, 177.93 mg C18:2-n6, 16.43 mg C18:3-n3.
Experimental design, animal management and diets
This study used eight lactating Saanen goats between the second and third lactation orders, with an average body weight (BW) of 54.30 ± 9.45 kg and milk production of 2.41 ± 0.56 kg, that were assigned in a replicate 4 × 4 Latin square. The experiment trial lasted for 76 days and was divided into four experimental periods with 14 days of adaptation to the experimental diets and five days of sampling and data collection. Goats were weighed at the beginning and end of each experimental period. Goats were distributed in individual pens (9.0 m2) with ad libitum access to feed and water.
The four experimental diets consisted of a control diet with no inclusion of CNSL, and inclusions of 5, 15 and 20 g of CNSL per kilogram of dry matter (DM). Before the daily feed offering, the cashew shell liquid (CNSL) was weighed based on each animal’s dry matter intake (DMI) to achieve the desired inclusion level. Subsequently, the CNSL was then added to the concentrate feed and homogenized manually. Subsequently, the concentrate was mixed with the roughage and offered to the animals as a total mixed ration. The diets consisted of 60% forage and 40% concentrate on a DM basis (Table 1).
Ingredient proportions, chemical composition and total fatty acid (FA) profile of total mix ration (TMR)
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM).
The diets were formulated with 16% of crude protein (CP) to meet the requirements for a 2.0 kg of milk production/day (NRC, 2007). The animals were fed twice daily (at 0800 and 1600 h). Before feeding the diet in the morning, refusals were collected and weighed. The DMI was determined as the difference between the amount of diet offered and the refusals and was adjusted to allow for up to 20% refusals. Feeds and refusals were sampled and frozen for subsequent chemical analysis.
Digestibility trial and nitrogen balance
The digestibility trial was carried out indirectly using indigestible neutral detergent fibre (iNDF) as to estimate faecal DM excretion, for that every 15 days faecal samples were collected from the rectal ampulla of the animals for three consecutive days at specific times (at 0800 h on the 1st day, noon on the 2nd day and 1600 h on the 3rd day) according to Pereira et al. (Reference Pereira, Pereira, Marcondes, Medeiros, Oliveira, Silva, Mizubuti, Campos, Heinzen, Veras, Bezerra and Araújo2018). At the end of the experimental period, a composite sample was mixed to be proportional to the amount excreted daily (DM basis). Posteriorly, faecal samples, refusals, concentrate and forage were incubated in situ throughout 240 h in the rumen of a cow receiving a diet of tifton hay and concentrate based on corn grain, soybean meal, limestone and dicalcium phosphate. The incubation was carried out using nylon bags with an approximate porosity of 50 µm at a rate of 15 mg/cm2 of sample per bag, according to the recommendation of Ørskov and McDonald (Reference Ørskov and McDonald1979). After the incubation period, the bags were washed in clean water until their colour was completely clear. Subsequently, the bags were boiled in a neutral detergent solution for 1 hour (Van Soest et al., Reference Van Soest, Robertson and Lewis1991), dried in an oven and the residues remaining after weighing were considered as iNDF. The total digestible nutrients (TDN) were calculated according to the methodology described by Sniffen et al. (Reference Sniffen, O'Connor, Van Soest, Fox and Russell1992).
Goat milk production and physical-chemical composition
The goats were milked twice daily (0700 and 1500 h). Milk production (weight and volume) was registered at every milking. On days 15 and 19 of each experimental period, milk production was measured, and samples were collected. For each goat, daily samples were prepared in proportion to the milk produced during each milking session (morning and afternoon) and stored in duplicate at −20 °C until further analysis. An aliquot of 10 ml milk was mixed with 5 ml of 25% trichloroacetic acid and filtered through filter paper to evaluate the urea content in the deproteinized milk. Another portion of milk was combined with a sodium azide solution (0.8%, wt/vol), used as a preservative (0.05 g/l), and subsequently lyophilized for subsequent FA analysis.
Urine collection, nitrogen balance and microbial protein synthesis
Spot urine samples were collected on the 15th day of each experimental period, approximately 4 hours after morning feed. Colostomy bags adapted to the goats’ vulva were used to collect urine through spontaneous urination. For the analysis of purine derivatives (PD), urine samples (5 ml) were collected and immediately diluted with 45 ml of a 0.018 mmol solution of sulfuric acid (H2SO4). Pure urine samples were used to calculate nitrogen balance. Both pure and diluted urine samples were stored at −20 °C until analysis. The concentration of creatinine in the urine was determined using a commercial assay kit (Labtest, Lagoa Santa, MG, Brazil), which was subsequently used to estimate urinary volume.
The nitrogen balance (g/d) was obtained by the difference between the total N-intake (g/d) and N-faecal, urinary and milk excretions (g/d). The nitrogen retained was expressed by the difference between nitrogen balance and basal endogenous nitrogen losses, considering the endogenous tissue and dermal N losses as 0.35 and 0.018 of metabolic weight, respectively (AFRC, 1993). Thus, basal endogenous nitrogen losses were estimated using the following equation:
\begin{equation}{\text{BEN }}\left( {{\text{g/day}}} \right){\text{ = }}\left( {{\text{0}}{\text{.018 + 0}}{\text{.35}}} \right){\times\, \text{B}}{{\text{W}}^{{\text{0}}{\text{.75}}}}\end{equation}where BEN corresponds to the basal endogenous nitrogen losses (g/d), and BW0.75 is metabolic body weight (kg).
Allantoin excretion was determined using the colorimetric method (Fujihara et al., Reference Fujihara, Ørskov, Reeds and Kyle1987). The uric acid excretion was determined using an enzymatic colorimetric test with clearing factor lipase (Barham and Trinder, Reference Barham and Trinder1972; Fossati et al., Reference Fossati, Prencipe and Berti1980). The intestinal flow of absorbed purine derivates and microbial nitrogen were estimated using equations proposed by Chen and Gomes (Reference Chen and Gomes1992):
\begin{equation}{\text{Y = 0}}{.84 \times \text{X + }}\left[ {{{\left( {{\text{0}}{\text{.150 BW}}} \right)}^{{\text{0}}{\text{.75}}}}{{\text{e}}^{{\text{( - 0}}{\text{.25X)}}}}} \right]\end{equation}where Y is expressed in mmol/day, X is the purine derivative absorption (mmol/d), 0.84 represents the recovery of absorbed purines as PD in urine, and the component within brackets represents the endogenous contribution, which decreases as exogenous purines become available for utilization by the goats.
\begin{equation}{\text{MN }}\left( {{\text{g/day}}} \right){ = 70 \times \text{ X/ }}\left( {{\text{0}}{\text{.116} \times \text{0}}{\text{.83} \times \text{1000}}} \right){\text{ = 0}}{\text{.727 X}}\end{equation}where MN is microbial nitrogen (g/d), assuming that the N concentration of purine is 70 mg N/mmol, the ratio of purine N to total N in mixed ruminal microbes is 11.6:100, and the digestibility of microbial purines is 0.83. The MN values were multiplied by a factor of 6.25 to obtain microbial CP (CPmic). The microbial protein synthesis (MPS) efficiency was estimated from the relationship between MPS (g/d) and TDN intake (kg/d).
Chemical analysis
Samples of feed, refusals and faeces were dried in a forced-air oven at 55ºC for 72 h and then ground in a Wiley mill (TE-650; Tecnal, Piracicaba, São Paulo, Brazil) with a 1-mm sieve. Subsequently, they were analysed for DM (method 967.03- AOAC, 1990), ether extract (EE; method 920.39-AOAC, 1990), ash (method 942.05-AOAC, 1990), CP (method 981.10-AOAC, 1990) and ADF (method 913.18-AOAC, 1990) according to procedures recommended by AOAC (1990). The NDF was measured with thermostable alpha amylase without sodium sulfite and corrected for residual ash (Mertens et al., Reference Mertens, Allen, Carmany, Clegg, Davidowicz, Drouches, Frank, Gambin, Garkie, Gildeimester, Jeffress, Jeon, Jones, Kaplan, Kim, Kobata, Main, Mona, Paul, Robertson, Taysom, Thiex, Williams and Wolf2002) and residual nitrogenous compounds (NDFap) (Licitra et al., Reference Licitra, Hernandez and Van Soest1996). The levels of total carbohydrates were obtained according to Sniffen et al. (Reference Sniffen, O'Connor, Van Soest, Fox and Russell1992), and non-fibre carbohydrates were calculated according to Weiss (Reference Weiss1993).
The nitrogen urea in milk was analysed using the Micro-Kjeldahl method, and the protein content was calculated as the nitrogen percentage multiplied by 6.38 based on method 991.20 (AOAC, 2005). The total dry extract from milk was obtained by drying in an oven at 105 °C until a constant weight was obtained according to method 925.23 (AOAC, 2005). The fat content in the milk samples was determined by the Gerber method (protocol 433/IV) and the content of lactose-reducing glycids (protocol 432/IV) according to the analytical standards of the Adolfo Lutz Institute (IAL, 2005). The acid activity was determined by method 947.05 (AOAC, 2005), where endpoint titration was based on lactic acid determined and expressed in Dornic degrees (°D).
Fatty acid analysis
To determine the milk FA profile, the milk samples were previously lyophilized and then used to prepare the FA methyl esters via direct transesterification using potassium hydroxide (2 M) in methanol, according to Rego et al. (Reference Rego, Alves, Antunes, Rosa, Alfaia, Prates, Cabrita, Fonseca and Bessa2009). Samples were analysed using a gas chromatograph (GC2010, Shimadzu®, Kyoto, Japan) equipped with a flame-ionization detector and a capillary column (SP2560, 100 m × 0.25 mm, df 0.20 μm thickness: Supelco®, Bellefonte, PA, USA). The column oven temperature was as follows: the initial temperature was maintained at 80 ºC, increased at 11 °C/min to 180 °C, and then at 5 °C/min to 220 °C, and then maintained for 19 min. The results were corrected with the theoretical correction factor of the flame ionization detector and expressed in mg/g of total FAs. The concentrations of SFA, UFA, MUFA, PUFA, ω-6 and ω-3 (n6 and n3, respectively) were calculated based on the milk FA profile. To determine the nutritional quality of the lipid fraction, the sum of the desirable FAs was calculated (Rhee, Reference Rhee and Chow2000). Then, the thrombogenicity index, the atherogenicity index (Ulbricht and Southgate, Reference Ulbricht and Southgate1991) and the ratio between the hypocholesterolemic and hypercholesterolemic FAs was determined (Santos-Silva et al., Reference Santos-Silva, Bessa and Santos-Silva2002). The activity of enzymes involved in lipid metabolism, such as Δ9 desaturase in C16, in C18 and elongase, was calculated according to the methods described by Malau-Aduli et al. (Reference Malau-Aduli, Siebert, Bottema and Pitchford1997).
Statistical analysis
The statistical analysis was performed using the SAS MIXED procedure. The statistical model used was:
\begin{equation}{Y_{ijkl}} = \mu + {T_i} + {S_j} + {P_k} + {A_l} + {e_{ijkl}}\end{equation}where Yijkl is the response variable measured in the animal; µ is the constant; Ti is the experimental treatments; Sj is the random effect of the square; Pk is the random effect of the period; Al is the random effect of the goat, and eijkl is the random error. Treatments were analysed as an orthogonal partition into linear and quadratic effects. A significance level of 5% (α = 0.05) was adopted in this study.
Results
Intake, digestibility and nitrogen balance
The DM (kg/d; %BW; g/kg0.75 BW), organic matter (kg/d), CP (kg/d), NDF (kg/d; %BW; g/kg0.75 BW), total carbohydrates (kg/d), nonfibrous carbohydrate (kg/d) and TDN (kg/d) intakes were not influenced (P > 0.05) by inclusions of CNSL. However, the EE intake (kg/d) increased linearly (P < 0.001) with an increasing inclusion level of CNSL (Table 2). The digestible coefficients of DM (P = 0.78), organic matter (P = 0.20), CP (P = 0.10), EE (P = 0.18) and NDF (P = 0.43) were not affected by the inclusion of CNSL (Table 3). There was no effect (P > 0.05) of CNSL on nitrogen balance (Table 4).
Effect of CNSL inclusions on nutrient and energy intake in lactating goats
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM); DMI, dry matter intake; OMI, organic matter intake; CPI, crude protein intake; EEI, ether extract intake; NDFI, neutral detergent fibre intake; NFCI, nonfibrous carbohydrate intake; TDNI, total digestible nutrients intake; DEI, digestible energy intake; MEI, metabolizable energy intake; SEM, standard error of the mean.
Effect of CNSL inclusions on nutrient digestibility in lactating goats
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM); DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; SEM, standard error of the mean.
Effect of CNSL inclusions on nitrogen balance in lactating goats
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM); NB, nitrogen balance; BEN, basal endogenous nitrogen losses; SEM, standard error of the mean.
Purine derivatives and milk composition
The inclusion of CNSL did not affect urinary volume (L/d), creatinine (mmol/d; mmol/kg BW/d; mmol/kg0.75 BW, uric acid and xanthine-hypoxanthine excretions (mmol/d) and MPS (g/kg TDNI) (P > 0.05) (Table 5). The CNSL inclusions did not influence (P > 0.05) milk yield (kg/d), FCM (kg/d), protein, fat and lactose (kg/d). The milk urea nitrogen (MUN, mg/dl) showed linear response (P < 0.001) by inclusion of CNSL in diets (Table 6).
Effects of inclusion of CNSL on purine derivatives (PD) in dairy goats
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM); AbsPD, absorbed microbial purine derivates; MPS, microbial protein synthesis; TDNI, total digestible nutrients intake; SEM, standard error of the mean.
Effects of inclusion of CNSL on milk yield and composition in lactating goats
a Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM).
b 4% fat corrected milk (FCM) estimated according to Gaines (Reference Gaines1927); FCM (kg/day), 0.432 × milk production (kg) + 0.1623 × milk fat concentration (g/100 g); MUN, milk urea nitrogen; SEM, standard error of the mean.
Milk fatty acid profile
Several FA peaks were detected; however, 22 individual FA were identified in goat's milk (Table 7). The inclusion of CNSL in diets did not affect (P > 0.05) C4:0, C6:0, C8:0, C10:0, C12:0, C13:0, C14:0, C14:1, C16:0, C16:1, C18:1-t11, C18:1-c9, C18:1-c11, C18:2-n6, C18:2-c9t11, C18:2-t10c12 and C20:4-n6. However, there was a linear response for C15:0 (P = 0.021), C17:0 (P = 0.029), C18:0 (P = 0.045), C18:1-t9 (P = 0.001) and C18:3-n3 (P = 0.007) by the inclusion of CNSL. Furthermore, a group of unidentified FA showed a linear increase (P < 0.001) with the inclusion of CNSL (Table 7).
Milk fatty acids profile (mg/g of total FA) in lactating goats fed with increasing levels of CNSL
Inclusion of 0, 5, 15 and 20 g of cashew nut shell liquid (CNSL, g/kg DM).
SEM, standard error of the mean.
The CNSL did not affect (P > 0.05) the partial sums of saturated (ΣSFA), unsaturated (ΣUFA), monounsaturated (ΣMUFA), polyunsaturated (ΣPUFA) and omega-6 (Σn6) FA. However, the partial sum of omega-3 (Σn3) FA showed a linear response (P = 0.007). The polyunsaturated/saturated (PUFA/SFA, P = 0.036), n6:n3 (P = 0.009) and n3:n6 FA (P = 0.005) ratios showed a linear response, and the PUFA/MUFA ratio decreased linearly (P = 0.035) with the inclusion level of CNSL. The UFA/SFA, MUFA/SFA and the ratio between the hypocholesterolemic and hypercholesterolemic FAs were not affected by the CNSL (P > 0.05). There was no effect (P > 0.05) of the CNSL on the thrombogenicity and atherogenicity indexes, and enzyme activities (Δ9 C16, Δ9 C18 and Elongase) (Table 8).
Partial sums of fatty acids (mg/g of total FA) ratios, indexes and enzyme activity on milk fat of lactating goats fed with increasing levels of CNSL
ΣSFA, partial sum of saturated FA; ΣUFA, partial sum of unsaturated FA; ΣMUFA, partial sum of monounsaturated FA; ΣPUFA, partial sum of polyunsaturated FA; Σn6, partial sum of omega 6 FA; Σn3, partial sum of omega 3 FA; P/S, Polyunsaturated/saturated FA ratio; U/S, unsaturated/saturated FA ratio; P/M, polyunsaturated/monounsaturated FA ratio; M/S, monounsaturated/saturated FA ratio; h/H, hypocholesterolemic/hypercholesterolemic FA ratio; AI, atherogenicity index; TI, thrombogenicity index; SEM, standard error of the mean.
Discussion
Our results confirm that CNSL can be provided as a dietary additive without negatively affecting performance and digestibility. The inclusion of CNSL in the diets did not restrict DMI, demonstrating that the amount of CNSL included in the diets did not cause toxic effects on the ruminal ecosystem. Several studies have demonstrated the toxic effect of CNSL on ruminal bacteria (Branco et al., Reference Branco, Giallongo, Frederick, Weeks, Oh and Hristov2015). The main compounds present in CNSL are anacardic acid, cardanol and cardol (Trevisan et al., Reference Trevisan, Pfundstein, Haubner, Wurtele, Spiegelhalder, Bartsch and Owen2006), these compounds are polyunsaturated alkylphenols, largely responsible for antimicrobial action, with a mechanism of action similar to ionophores (Nagabhushana et al., Reference Nagabhushana, Shobha and Ravindranath1995). They increase the fluidity of the cell membrane of ruminal microorganisms, resulting in the loss of selective permeability and reducing cell viability (Rodrigues et al., Reference Rodrigues, Feitosa, Ricardo, França and Carioca2006). This antimicrobial action could lead to reductions in ruminal fermentation and consequently a decrease in DMI (Shinkai et al., Reference Shinkai, Enishi, Mitsumori, Higuchi, Kobayashi, Takenaka, Nagashima, Mochizuki, Kobayashi and Mochizuki2012; Araújo et al., Reference Araújo, Araújo, Silva, Pereira Filho, Parente, Oliveira, Mazzetto, Oliveira, Edvan and Bezerra2023), which did not occur in our study.
Ether extract intake, as expected, increased as CNSL was included in the goats’ diets, as this additive has a high lipid concentration. However, dietary extract levels remained close to the recommended levels (2–4% of total dietary DM) to avoid deleterious effects on fibre degradation (Jenkins, Reference Jenkins1994), as confirmed in this study, as there were no significant effects of CNSL inclusion on nutrient digestibility.
In our study, the purine derivative excretion and MPS were not affected by the inclusion of CNSL. MPS is relatively dependent on the availability of carbohydrates and nitrogen in the rumen (NRC, 2001). For successful MPS, both nitrogen sources and degradable carbohydrates must be synchronized in quality and degradation rate (Chen and Gomes, Reference Chen and Gomes1992). The lack of significant effect of the inclusion of CNSL on the MPS indicates that the inclusion levels adopted in this study did not negatively affect ruminal fermentation and microbial activity. In addition, rumen fermentation can be monitored by the milk urea nitrogen content due to its response to the efficiency of microbial protein (Broderick and Clayton, Reference Broderick and Clayton1997) and the adequacy of dietary protein: energy ratio levels (Bittante, Reference Bittante2022). In our study, MUN excretion (mg/dl) was lower for animals fed a diet containing 5 g CNSL/kg DM, and the values of MUN are within the range described by Jonker et al. (Reference Jonker, Kohn and Erdman1999) of 10–16 mg/dl, which indicates a synchronization of nitrogen compounds and energy sources. Parameters such as DMI, digestibility, and milk production and composition are important in the selection and application of new additives, as they are related to productive performance and the modulation of ruminal fermentation. Therefore, the absence of negative effects of CNSL on these parameters may be a key factor in its acceptance as an additive in production systems.
Milk fat plays an important role in determining the quality and physical properties of dairy products (Teng et al., Reference Teng, Wang, Yang, Ma and Day2017). Furthermore, the flavour of goat’s milk is highly correlated with its lipid content and lipolysis (Pereira et al., Reference Pereira, Oliveira, Medeiros, Costa, Bomfim and Queiroga2010). In this sense, it is important to consider that the end consumer’s perception of a product is based on the perception of different attributes that constitute its sensory quality, and the perceived sensations depend on the intensity and interactions between these attributes (Pereira et al., Reference Pereira, Oliveira, Medeiros, Costa, Bomfim and Queiroga2010). Therefore, providing diets that can improve the quality of nutraceutical compounds without affecting their sensory attributes can aid in the acceptance of goat’s milk (Morais et al., Reference Morais, Bezerra, Silva, Araújo, Oliveira, Edvan, Torreão and Lanna2017). In our study, the amount of milk fat was not affected by the inclusion of CNSL; therefore, even though it was not the main objective of this study, we believe that the quality of the sensory attributes was maintained at all levels of inclusion, given the antioxidant capacity of CNSL (Trevisan et al., Reference Trevisan, Pfundstein, Haubner, Wurtele, Spiegelhalder, Bartsch and Owen2006).
The fat composition of milk plays an important role in determining the quality and physical properties of dairy products (Teng et al., Reference Teng, Wang, Yang, Ma and Day2017). Additionally, it is crucial for animal health, as nutrient imbalances can alter microbial populations, subsequently affecting the composition of FAs (Carta et al., Reference Carta, Correddu, Battacone, Pulina and Nudda2022).
In this study, CNSL affected C15:0 and C17:0 goat milk. Differences in the FA profile of goat milk may indicate changes in rumen microbiota (Vlaeminck et al., Reference Vlaeminck, Gervais, Rahman, Gadeyne, Gorniak, Doreau and Fievez2015). Thus, a change in the ruminal microbiome due to the inclusion of CNSL may have influenced the synthesis of linear-chain FAs such as 15:0 and 17:0. CNSL contains a high amount of anacardic acid, a phenolic compound that is an antimicrobial with selective activity on ruminal microorganisms, leading to fermentation patterns that result in increased propionate production and reduced methane production (Watanabe et al., Reference Watanabe, Suzuki, Koike, Nagasima, Mochizuki, Forster and Kobayashi2010; Shinkai et al., Reference Shinkai, Enishi, Mitsumori, Higuchi, Kobayashi, Takenaka, Nagashima, Mochizuki, Kobayashi and Mochizuki2012). Studies have shown that linear-chain FAs such as 15:0 and 17:0 can be synthesized de novo using propionyl-CoA instead of acetyl-CoA as the initiator for FA synthesis in the mammary gland (Massart-Leën et al., Reference Massart-Leën, Roets, Peeters and Verbeke1983; Vlaeminck et al., Reference Vlaeminck, Gervais, Rahman, Gadeyne, Gorniak, Doreau and Fievez2015). Thus, we suspect that the inclusion of CNSL may have modified the ruminal microbiota, increasing the population of bacteria related to propionate production, favouring the increase of these FAs in the goats’ milk.
The dietary inclusion of CNSL influenced the concentrations of stearic acid (C18:0). Although classified as a saturated fatty acid (SFA), stearic acid is endogenously desaturated to oleic acid (C18:1), a monounsaturated FA associated with beneficial health effects and considered neutral with respect to plasma cholesterol levels (Gama et al., Reference Gama, Pereira Filho, Soares, Cordão, Cézar, Batista, Silva, Madruga, Oliveira and Bezerra2020). This outcome may be attributed to the low dietary intake of these specific FAs and the unaltered lipid composition of milk, as the experimental diets did not affect total milk fat content. Additionally, CNSL supplementation did not modulate the activity of lipogenic enzymes responsible for the synthesis of short- and medium-chain FAs (Kim et al., Reference Kim, Adesogan, Badinga and Staples2007). Therefore, milk produced by goats fed CNSL can be considered nutritionally favourable, as it does not exhibit elevated levels of hypercholesterolemic FAs, particularly palmitic acid (C16:0) and myristic acid (C14:0).
In this study, the linear increase in C18:1-t9 FA as CNSL inclusion increased in the diet can also be explained primarily by changes in the ruminal microbiota due to the antimicrobial action of CNSL (Shinkai et al., Reference Shinkai, Enishi, Mitsumori, Higuchi, Kobayashi, Takenaka, Nagashima, Mochizuki, Kobayashi and Mochizuki2012). Some studies have demonstrated a correlation between the production of some FAs and the rumen microbiota (Buitenhuis et al., Reference Buitenhuis, Lassen, Noel, Plichta, Sørensen, Difford and Poulsen2019; Liu et al., Reference Liu, Zhang, Huang, Zheng, Zhao and Wang2022). Si et al. (Reference Si, Liu, Huang, Chen, Yang, Wu, Li, Tang, Zhao, Zheng, Zhang and Wang2023) identified a relationship between some genera of ruminal bacteria, such as Prevotella, that promote the deposition of FAs such as C18:1-t9. Shinkai et al. (Reference Shinkai, Enishi, Mitsumori, Higuchi, Kobayashi, Takenaka, Nagashima, Mochizuki, Kobayashi and Mochizuki2012) reported that bacteria of this genus increased as CNSL was included in the diet of dairy cows. Thus, we suspect that the change in the ruminal microbiome provided the increase in this FA.
The C18:2-c9 t11 and C18:2-t10 c12 are the most important natural isomers of CLA due to their biological activity (Kennedy et al., Reference Kennedy, Martinez, Schmidt, Mandrup, LaPoint and McIntosh2010). In our study, CNSL levels were not sufficient to express an influence on the secretion of both CLA isomers. The rumenic acid presents anticarcinogenic properties (Jensen and Lammi-Keefe, Reference Jensen, Lammi-Keefe and Newburg2001) in addition to positively influencing on muscle growth (Shahidi and Senanayake, Reference Shahidi, Senanayake and Heggenhougen2008), while the C18:2-t10 c12 isomer is correlated with a reduction in milk fat (about 78%), inhibiting lipid metabolism, de novo synthesis in the mammary gland and reducing the percentage of short- and medium-chain FA (Baumgard et al., Reference Baumgard, Corl, Dwyer, Saebø and Bauman2000, Reference Baumgard, Matitashvilli, Corl, Dwyer and Bauman2002).
The inclusion of CNLS influenced the ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratio; however, the observed values remained elevated. According to Wood et al. (Reference Wood, Enser, Fisher, Nute, Sheard, Richardson and Whittington2008), a ratio below 4.0 is considered optimal for the human diet in order to reduce the risk of cardiovascular diseases. The increased ratio observed is likely attributable to changes in the concentrations of linoleic acid and α-linolenic acid, which are the principal representatives of ω-6 and ω-3 PUFAs, respectively. In this study, we observed that the goats receiving 5 g of CNSL/kg DM in the diet excreted higher C18:3 n-3 (4.36 mg/g total FA) in milk. This result is directly proportional to the levels of this FA in diets. Diets rich in polyunsaturated fatty acids may be used as a nutritional strategy to increase milk PUFA content (Pérez et al., Reference Pérez, Padilla, Mendoza, Morales, Leskinen, Garnsworthy, Loor and Romero2020), since they are considered essential because they are not synthesized in the animal’s body (Chilliard et al., Reference Chilliard, Ferlay, Rouel and Lamberet2003). The α-linolenic acid (C18:3 n-3) is a precursor of n-3 PUFA such as eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3). These FA have an important role in metabolic and inflammatory processes (Serhan and Chiang, Reference Serhan and Chiang2008), insulin sensitivity and glucose homeostasis (Flachs et al., Reference Flachs, Rossmeisl and Kopecky2014), and in the prevention of coronary heart disease (Póti et al., Reference Póti, Pajor, Bodnár, Penksza and Köles2015) and diabetes mellitus (Muley et al., Reference Muley, Muley and Shah2014).
Conclusions
The inclusion of CNSL in the diet of lactating goats did not affect nutrient intake, digestibility, xanthine-hypoxanthine excretions and MPS (g/kg TDNI), milk yield (kg/d), protein, fat, and lactose (kg/d), but increased milk urea nitrogen, C15:0, C17:0, C18:1-t9 and C18:3-n3 in milk. The CNSL did not alter the partial sums of saturated (ΣSFA), unsaturated (ΣUFA), monounsaturated (ΣMUFA), polyunsaturated (ΣPUFA), and omega-6 (Σn6) FAs; however, the partial sum of omega-3 FAs (Σn3) was increased. The CNSL is a potential additive for use.
Acknowledgements
The authors thank the grants provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES and Institutos Nacionais de Ciência e Tecnologia INCT – Ciência Animal and Cadeia Produtiva da Carne.
