Ethical statement

This research complied with the requirements of the Research and Postgraduate Secretary of the Autonomous University of Nayarit, with registration SIP15-65.

Animals and diets

Twenty-four castrated Landrace-Yorkshire pigs with an initial weight of 55 ± 3 kg were used, housing one pig per pen with individual access to food and water. Pigs were fed ad libitum, according to the recommendations of the official Mexican animal welfare norm (NOM-062-ZOO-1999). After five days of adaptation to the diets, plus 56 days of experimental study, the pigs were sacrificed by a method approved by national regulation contained in NOM-033-SAG/ZOO-2014 (Lightheadedness followed by cutting of caval veins and brachiocephalic trunk), at an average live weight of 109 ± 4 kg. Eight pigs were assigned to each diet. The diets had 0, 5 and 10 % avocado meal (AM) (Tables 1, 2). The AM was obtained as described⁹ and the proximal chemical characteristics and fatty acid profile have been reported.¹¹ Hass avocados discarded for human consumption due to their small size and/or physical damage were used in the diets. The fruits were stored at room temperature until they reached maturity. To obtain a homogeneous mixture of ripe and whole avocados (pulp, seed, and shell), the fruit was ground in a mobile hammer mill without a sieve, driven by a 5 HP gasoline engine. The fresh paste was stored at room temperature without additives, in plastic containers, and then, it was left at room temperature for four days until was dried. Then, the dry paste was ground again to incorporate it into the pig diets.

Analysis of transcriptome on in the L. dorsi muscle and liver

Three pig RNA samples were considered per experimental diet for L. dorsi muscle and liver, (n = 18 total samples) and 75 mg of each tissue per pig was taken for RNA extraction. RNA extraction was performed using the nucleic acid extraction kit Direct-zol™ RNA MiniPrep (Zymo Research, USA); whereas, the RNA concentration and purity were quantified by spectrophotometry with a Nanodrop. Massive sequencing was carried out in the UUSMB-UNAM (University Mass Sequencing and Bioinformatics Unit) sequencing service, with Illumina methodology, on a Nextseq 500 device (www.illumina.com/company/legal.html), pared (pair end) of 76 bp, from 18 pork samples, with average quality above Q28 for all samples in all cycles. The sequences obtained were mapped against the Sscrofa11.1 pig reference genome using the Smalt 0.7.6 program (https://bioweb.pasteur.fr/packages/pack@smalt@0.7.6). The per gene counts were carried out using the Bamtools (https://bio.tools/bamtools), coverage bed program (27 280 records), a script in Perl to filter exclusively coding genes for proteins (16 841 records) with final a reading of 15 760 records. With these records, differential expression analysis of L. dorsi muscle and liver, for AM5 vs AM0 and AM10 vs AM0 was done. IDEAmex software (http://www.uusmb.unam.mx/ideamex/),¹² was used to apply the EdgeR and DESeq2 methods, and obtain differentially gene expression (DGE), with a reliability statistic of P ≤ 0.01 and a log2 fold change ≥ 1.5.

Growth and carcass traits

At the end of the experimental period, average daily gain (kg), average daily feed intake (kg), feed/gain ratio, slaughter weight (kg), hind carcass weight (kg) without viscera and skin, carcass (%), ham (kg) and back fat (mm) were registered.

Intramuscular fatty acids profile of L. dorsi muscle

After the slaughter of pigs, 100-g L. dorsi muscle samples were collected, and the lipid composition was determined according to the method 920.39.¹³ To extract L. dorsi muscle lipids, 500 mg of fat was converted into methyl esters fatty acids using chromatographic analysis.

Blood metabolites in finishing pigs

At slaughter, blood samples were obtained from pigs for biochemical assays, which were carried out using Byosistem A160 spectrophotometry equipment. Glucose (mg/dL). Lipid profile: Total cholesterol (mg/dL), triglycerides (mg/dL), HDL (high density cholesterol) (mg/dL), LDL (low density cholesterol) (mg/dL), VLDL (very low density cholesterol) (mg/dL) and HDL/LDL ratio. Liver profile: Urea (mg/dL), Uric acid (mg/dL), R A/G (Albumin/Globulin Ratio), TGO (oxalacetic glutamic transaminase) (mg/dL), TGP (pyruvic glutamic transaminase) (mg/dL) and GGT (gamma-glutamil transferase) (mg/dL) were also measured.

Statistical analysis

The effect of diet on growth, carcass, L. dorsi muscle, fatty acid composition and blood metabolites was determined using one-way analysis of variance under a completely random experimental design, and mean comparisons by Tukey test. Additionally, linear and quadratic regression analyses of the levels of inclusion of AM and measured variables were carried out. Gene annotation: biological function using the NCBI database and Ensembl pig genome databases (http://uswest.ensembl.org/Sus_scrofa/Info/Index), ShinyGO v0.61: Gene Ontology Enrichment Analysis (http://bioinformatics.sdstate.edu/go/) and PHANTER GO (http://geneontology.org/) were searched.

Transcriptome analysis of the L. dorsi muscle and liver

The transcriptome analysis revealed higher DGE for AM10 vs AM0 on L. dorsi muscle and liver (Table 3), and higher DGE in both tissues, with down log₂ fold change by including 5 or 10 % AM in the diet. Furthermore, higher DGE in both tissues was observed in chromosomes (Chr) 6 and 2, but unidentified in Chr 11 (Figure 1).

Figure 1 Identification of genes by tissue on different chromosomes. 

Tables 4 to 7 show higher DGE in L. dorsi muscle and liver when AM5 and AM10 diets were compared with the control diet. Higher DGE in AM10 was observed in the anatomical structure development and morphogenesis of L. dorsi muscle in, as was the effect identified in the biological processes: regulation of cellular component biogenesis and cell proliferation, regulation of the immune system process and system development, response to stress and cellular response, including lipid metabolism. With respect to the liver, AM5 and AM10 diets had higher DGE with down expression in anatomical structure development and morphogenesis. However, comparation of AM5 and AM0 diets, identified down DGE in liver biological process: Lipid metabolism, negative regulation of growth, response to stress and cellular response. AM10 and AM0 diets comparison showed down DGE for negative regulation of growth, regulation of cellular component biogenesis and cell proliferation, regulation of the immune system process and system development, response to external stimuli, response to stress and cellular response; in addition, to transport of fatty acids or hormones.

Eleven genes were identified with DGE in more than one comparison (Table 8). Stand out genes associated with biological processes related to growth, response to stress, lipid metabolism and circadian rhythms with down expression, most of them in the liver. The ARNTL gene was up expressed in both tissues evaluated in pigs fed the AM10 diet, associated with anatomical structure development and morphogenesis, as well as circadian rhythms of locomotor activity and behavior.

Growth, carcass, L. dorsi muscle, fatty acid composition and blood metabolites

The effect of diets on growth, carcass, L. dorsi muscle fatty acid composition and blood metabolites are shown in Tables 9, 10, and 11, respectively. Including 5 and 10 % AM in the diet increased daily gain, daily feed intake, slaughtered body weight, carcass weight and ham. The highest back fat mean was observed for the AM10 diet. All measured traits increased with AM, present significant quadratic regression effects. The AM diets had a negative linear regression effect on IMF. The AM10 diet had the highest values of linoleic acid, total fatty acids Ω6, ∑ polyunsaturated fatty acids, PUFA/SFA, and PUFA/MUFA; all variables with a positive linear regression effect. The inclusion of AM in the diet did not affect glucose, total cholesterol, lipoproteins, metabolites, or enzymes of liver function. Interestingly, while the 5 % AM diet decreased the triglycerides and VLDL content, the AM10 diet did not.

Transcriptome analysis in L. dorsi muscle and liver

The inclusions of 5 or 10 % of AM in the diet caused higher DGE in L. dorsi muscle and liver, identified with down log₂ fold change. It is considered that the liver plays an important role in the lipid metabolism of IMF.¹⁴ However, L. dorsi muscle also had a high number of DGE. This effect is observed in the decrease in IMF and in the changes in fatty acids of pigs fed the AM10 diet. In both tissues, higher DGE was identified in chromosomes 6 and 2 of pigs on AM5 and AM10 diets.

The above referred chromosomes are involved in growth and meat quality in pigs (https://www.animalgenome.org/cgi-bin/QTLdb/SS/browse). With top DGE and down log₂ fold, comparison of AM5 or AM10 vs AM0 diets identified the ANO3 gene, located in Chr 2, which is involved in lipid metabolism, associated in this study with a decrease in IMF. The down DGE of the PHGDH gene in L. dorsi muscle, involved in muscle growth, and in the liver,¹⁵ the down DGE of LOC100739663, LOC102166944, MT1A, and MT1D genes, involved in the negative regulation of growth, could be associated with the AM10 diet. Moreover, slaughter weight, hind carcass weight and ham decreased in the AM5 diet. Other DGEs with up expression in L. dorsi muscle involved in lipid metabolism were GOS2 in AM5 vs AM0, and MYLIP in AM10 vs AM0 diets; and in the liver the PLIN4 gene with AM10 vs AM0.

The up expression of the GOS2 gene in L. dorsi muscle from Laiwu pigs, was associated with fat metabolism and changes in fatty acids.⁶ The MYLIP gene that regulates myosin, LDL and VLDL, acts as a sterol-dependent inhibitor of cellular cholesterol uptake by mediating ubiquitination and subsequent degradation of LDL, and may be associated with the low value of LDL in AM10. The PLIN4 gene located in Chr 2 is strongly associated with fatty acid composition in the Longissimus thoracis muscle of Iberian pigs,¹⁶ which could influence the AM10 diet to have more changes in the fatty acid profile. Important genes related to anatomical structure development and morphogenesis were identified here, showing a higher down DGE in the AM10 diet, in L. dorsi muscle and liver. The ARNTL gene located in Chr 2 showed up DGE in both tissues (AM10 vs AM0 diet).

Other auhors found that this gene is a master regulator in the Alentejano obese breed,¹⁷ being activated and involved in the proliferation of fibroblast cell lines and the activation of myeloid cells. This is an indication of connective tissue development and an active state of the innate immune system, probably due to inflammation associated with obesity. The ARNTL gene could be associated with the results obtained when feeding AM10, since backfat was increased.

We found a down expression of the FOS gene in the Chinese Debao pig compared with Landrace, which influenced the pork quality between those breeds, being better for Debao pigs.¹⁸ The FOS gene, which is associated with the differentiation of adipocytes, in this work, had a down DGE in L. dorsi muscle (AM10 vs AM0 diet), as well as in the other 21 genes identified. The genes PER1, PER2, PER3, and SIK1 that are important for circadian rhythms of locomotor activity and behavior,¹⁹ showed down DGE in L. dorsi muscle for the AM10 diet vs AM0; and in the liver, the SIK1 gene (AM5 vs AM0), and PER1, NOCT, and DBP genes (AM10 vs AM0).

The ARNTL gene, which is associated with the circadian clock, had the opposite result: up DGE in L. dorsi muscle and liver for AM10 diet, but did not affect the circadian clock. In the pig muscle, nutrition modulates the circadian clock and those genes change their expression in response to nutrient intake.¹⁹ These authors reported that in pigs given food after fasting, the expression of ARNTL gene increased and the expression of PER1, PER2, PER3, SIK1 decreased. The ARNTL gene is a regulator of the circadian clock repressor genes PER1, PER2, and PER3, whose expression decreases due to environmental effects, while increasing that of repressor genes,²⁰ which did not happen for AM10.

The NOCT gene is also associated with the circadian clock as it promotes adipogenesis and resistance to high-fat diets when knockout.²¹ Therefore, in the AM10 diet, the NOCT gene is down expressed, which could favor an increase in backfat. Higher down DGE for regulation of the immune system process were identified by feeding AM10 vs AM0 in both tissues. Within those, OTUD1, CD163 and CRP genes had similar reports where the low expression was favorable to the animal. The OTUD1 gene is a crucial negative regulator of innate antiviral immunity, in knockout cells and mice, where more resistance to lethal infections is reported.²² The CD163 gene in knockout pigs is fully resistant to high-pathogenic porcine reproductive and respiratory syndrome virus.²³ The CRP gene also increases its levels as an indicator of inflammatory processes.²⁴

Other up DGE elements, favorable for regulating the immune system process, identified when animals were fed AM10 and AM0 diets were C1QTNF7 gene, related to the immune response; HP, an antioxidative molecule that prevents the hemoglobin driven generation of hydroxyl radicals and lipid peroxides; NCF4 superoxide-generating NAD(P)H oxidase activity, and NFIL3 involve in the cellular response to interleukin-4 (www.ensembl.org/Sus_scrofa/Gene/Summary).²⁵

Growth, carcass, L. dorsi muscle, fatty acid composition and blood metabolites

Feeding pigs before slaughter improved feed consumption when AM in the diet was included. This increased the daily consumption of metabolizable energy, oleic, linoleic and linolic fatty acids since AM contains a high amount of these monounsaturated and polyunsaturated fatty acids. However, the inclusion of 5 to 10 % AM in the diet showed a quadratic regression effect suggests a tendency to decrease as the inclusion of AM increases, which limits the increase in the levels of inclusion of AM in the diets. This could be due to the high content of tannins in AM that could have depressed feed consumption.^11,26 Therefore, although the growth and carcass of pigs increased from 0 to 5 % AM the rate of improvement was less o declined when 10 % AM was fed. A different situation was found with diets rich in sunflower in Duroc pigs, where no effects on growth and fattening traits were found.^3,4 However, sunflower affected the constitution of fatty acids with higher PUFA and the activation of the de novo lipid synthesis, as observed here in pigs fed AM.

Feeding AM5 and AM10 increased the average daily gain, slaughtered body weight, hind carcass weight and ham; with higher back fat and lower IMF in L. dorsi in the AM10 diet. All these variables increased with dietary AM, with a significant quadratic regression effect, suggesting a tendency to decrease as the inclusion of AM increases; caused by the fact that the consumption of AM does not have a linear effect, which limits the nutrients available for growth. Our results for growth and carcass traits were higher than those reported for Yorkshire and Duroc,^3,27 where a linear decrease in IMF was observed.

The pigs fed the AM10 diet had higher values of linoleic acid, total fatty acid Ω6, ∑ polyunsaturated fatty acids, PUFA/SFA and PUFA/MUFA, which is suggestive of a greater activation of the de novo lipid synthesis with this diet. Those traits had a positive linear regression effect, although the feed consumption was not linear. This trend indicates that the greater nutrient content in the AM10 vs AM5 diets (i.e. Mcal/kg ME, oleic, linoleic and arachidonic) influenced results, which is further supported by the fact that there is a greater amount of tocopherol and antioxidants in the AM.^8,9 Therefore, it is reasonable to conclude that the profile of fatty acids in adipose tissue could be modified by the source of lipids since similar results for MUFA and PUFA were reported²⁸ in pigs fed diets with 10 % sunflower oil, linseed oil, or a combination of fish oil-linseed.

Additionally, including avocado paste in the diet of Yorkshire-Landrace pigs was reported to increase PUFA in the L. dorsi muscle and decrease IMF.²⁹ These authors concluded that breed differences exist between Chinese and Yorkshire pigs in the fatty acids,²⁷ in accordance with previous studies.^3,4,7,30 This experimental evidence agrees with the fact that local pig breeds had a higher content of MUFA and lower PUFA, and that both could be modified with lipid sources in the diet of commercial pig breeds.²

In this study, the inclusion of AM5 and AM10 in the diet did not affect MUFA content, but the decrease in IMF with higher PUFA is possibly associated with a decrease in triglycerides. According to investigations,^7,31 the balance between synthesis, degradation and uptake of triglycerides is reflected in IMF content as intramuscular triglycerides are not only stored in the adipocytes but also as droplets in the myofiber cytoplasm. It has been reported that a high glycolytic activity reduces the deposition of IMF.³¹ This phenomenom could explain the increase of muscle deposition in pigs fed the AM10 diet at the expense of a lower IMF content. IMF fatty acid composition is a complex polygenic trait whose variability depends on feeding aspects and the genetic background of the pig population^1,2,6,17. It is necessary to identify the factors that affect the composition of the fatty acids in meat, to find a balance between the demands of consumers and the industry.