**3. Results**

### *3.1. Meat Quality Traits*

Meat quality traits evaluated in the *longissimus lumborum* (LL) muscle of fattening pigs fed with different levels of SWA are presented in Table 2. The pH values (measured 45 min and 24 h *postmortem*) for every treatment were in the range of normal values without being statistically different among treatments (*p* > 0.05). The ANOVA detected a significant effect of treatment (*p* = 0.05) on the a value (redness). Meat samples from the 4%-SWA treatment had less red intensity values when compared to those from the 2%-SWA and the control group (*p* ≤ 0.05). No difference (*p* > 0.05) was observed for L value (lightness) and b value (yellowness). The instrumental tenderness and the cooking losses (expressed in percentage) were not affected by the SWA treatments (*p* > 0.05). The samples from the control group and 4%-SWA resulted in the best taste preference by panelists, being significantly different from the sample of the 2%-SWA group (*p* ≤ 0.05). Samples from the control and the 4%-SWA groups showed a similar percentage of taste preference (Figure S1).


**Table 2.** Effects of the inclusion of seaweed additive (SWA) in the diet of fattening pigs on meat quality traits.

Means within a row lacking a common superscript letter differ (*p* ≤ 0.05). SEM: Standard Error Mean.

### *3.2. Nutrient Composition of Meat*

The ANOVA revealed that the SWA treatments only affected the total content of ash (*p* < 0.001). The LL samples of animals that were fed with the 4%-SWA presented a higher percentage of total ash compared to the other treatments (Table 3; *p* ≤ 0.05). However, no mean differences in the total ash content were found (*p* > 0.05) when comparing the control with the 2%-SWA group. The LL samples from the 4%-WSA exhibited a decreased percentage of total lipids without being statistically different (*p* = 0.07).


**Table 3.** Effects of the inclusion of seaweed additive (SWA) in the diet of fattening pigs on the proximate composition of pork meat.

1 values are expressed as g/100 g of fresh muscular tissue. Means within a row lacking a common superscript letter differ (*p* ≤ 0.05). SEM: Standard Error Mean.

The ANOVA showed a significant effect of the SWA treatments on the total cholesterol content (*p* ≤ 0.05). Figure 1 shows that meat samples from animals fed with the highest % of SWA (4%) contained 3.37 and 3.81 mg/100 g more muscular tissue cholesterol content than the control and the 2%-SWA groups, respectively (*p* ≤ 0.05).

**Figure 1.** Mean values ± standard error means of total cholesterol in pork meat according to SWA treatments (*p* = 0.02). The different superscript letters represent significant statistical differences (*p* ≤ 0.05).

The ANOVA showed that the SWA inclusion in the diet of fattening pigs did not affect the composition of saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) detected in the pork meat samples of this study. The ANOVA also found that the SWA treatments did not affect (*p* > 0.05) any of the health indexes associated with fatty acids composition: the ratio hypercholesterolemic index/hypocholesterolemic index, atherogenic index, thrombogenic index. In supplementary material (Table S1) are shown the descriptive statistics for fatty acids composition and health index in pork LL muscles.

Table 4 shows the mean values and standard error mean of several minerals evaluated in the LL samples of this study. The SWA treatments affected (*p* ≤ 0.05) the content of the micro-minerals Mn, Fe, and Cu. The samples of meat with the greatest percentage of SWA (4%) had 0.13, 0.45, and 0.23 less mg/100 g of muscle in Mn, Fe, and Zn, respectively, compared with the control group (*p* ≤ 0.05). The content of minerals of Na Mg and Zn were not different (*p* > 0.05) among the treatment groups; however, there was a trend (*p* = 0.08) of meat samples from the 4%-WSA treatment containing an inferior amount of K when compared to the 2%SWA and the control groups.

**Table 4.** Effects of the inclusion of seaweed additive (SWA) in the diet of fattening pigs on the mineral content in pork meat.


1 values are expressed as mg/100 g of fresh muscular tissue. Means within a row lacking a common superscript letter differ (*p* ≤ 0.05). SEM: Standard Error Mean.

By expressing the proximal composition of the meat on a dry basis (DM), the highly significant effect on the ash content was confirmed (Table 5; *p* < 0.0001). The samples from the animals that consumed the highest amount of SWA administered in the pigs' diet generated an increase in the amount of total ash DM of the meat. The Mn and Cu content decreased (*p* = 0.002) in the treatment with 4%-SWA, compared to control samples, but they were statistically similar to those of the 2%-SWA treatment. In addition, a trend could be evidenced in the results obtained in the Fe content (*p* = 0.06), where meat samples from 2%-SWA exhibited the lowest content of this mineral.

**Table 5.** Means of proximate composition and mineral content based on dry matter (DM) by treatments.


1 values expressed as g/100 g of DM. 2 values expressed as mg/kg of DM. Means within a row lacking a common superscript letter differ (*p* ≤ 0.05). SEM: Standard Error Mean.

### **4. Discussion**

The effects of the inclusion of additives or supplements based on brown seaweed have been tested on growth performance, nutrients digestibility, prebiotic, antioxidant, anti-inflammatory, and immunomodulatory activities in pigs [21–24]; however, the impact

of these dietary interventions on pork meat quality and nutritional composition has been less studied.

The main traits that define pork quality are pH, color, and water holding capacity [36]. The *postmortem* pH variation is an important factor that determines meat quality and has an influence on the physicochemical traits and shelf life [37,38]. Former investigations had found that the addition of seaweed in the animal diet could change carcass characteristics like marbling, color, and pH in pork [25–27,39]. Muscle pH values in this study were not affected by the inclusion of SWA and were ranged in the normal values. Muscular pH is highly correlated with the energy content of the diet [40]; however, the rate of pH decrease is influenced by multiple *antemortem* factors [41] and *postmortem* manipulation [38,42]. Michalak et al. [22] reported no statistically significant effect of a green seaweed additive (*Enteromorpha* sp.) on pH, water capacity holding, and drip loss in pork meat. Rossi et al. [19] found that sensory traits like aroma, flavor, and aroma of rabbit meat were affected by the use of 0.3 and 0.6% of dietary brown seaweed in the diet (*Laminaria* sp.). In this study, a better taste preference was observed from pork samples of animals that were fed with the highest % of SWA, without being different from those from the control groups (Figure S1).

According to the pork meat color standard [43], lightness values between 37 and 49 are considered normal. In this study, L values were not affected by treatments, and their values ranged from 44.95 to 47.61. Brown seaweed has been reported to be a rich source of natural antioxidants such as polysaccharides and polyphenols, which could improve meat color display [28,44]; also, antioxidant, antimicrobial, and immunomodulatory activities have been reported for compounds (extracts) of brown algae [8]. Moroney et al. [25] reported that spray-dried seaweed extracts that contained laminarin and fucoidan did not affect the redness values when incorporated in fresh pork via the animal diet. However, in another study, the same authors [26] reported that the SWA significantly reduced the redness intensity compared with the control. Additionally, Rajauria et al. [28] reported that an addition of 5.3% of seaweed extracts (*Laminaria* spp.) in the diet of finishing pigs significantly reduced the redness intensity compared to the control. In this study, the redness intensity was also significantly reduced in samples from animals that were fed with the highest percentage of SWA (4%).

The lower values detected in meat samples from animals that were fed with the highest % of SWA in this study could have been related to the lower iron content present in the muscle from the same treatment (4%-SWA), or could also be related to some interactions between the polysaccharides present in the SWA and the oxymyoglobin in the pork meat [14].

In this study, meat samples from 4%-SWA treatments had lower levels of iron. Ponnampalam et al. [45] reported that increased muscle heme iron concentration resulted in higher values in beef displayed for 48 to 72 h *postmortem*.

We hypothesized that samples from pigs that were fed with the highest % of SWA would increase the PUFA values and reduced cholesterol content; however, meat samples from animals that were fed a regular diet plus 4% SWA go<sup>t</sup> the highest levels of total cholesterol compared to the other treatments (*p* < 0.05). Supplementation of laying hens with 1–2% of dried *Enteromorpha porifera* seaweed resulted in a reduction in cholesterol in the yolk [18]. Rossi et al. [19] reported that the inclusion of dietary levels of brown seaweed (*Laminaria* spp.) did not affect the content of cholesterol in rabbit meat. To our knowledge, there is no previous report on the effect of dietary SWA on cholesterol content in pork samples. Our results sugges<sup>t</sup> that there is an important effect of the components of seaweed that impact the content of cholesterol. Muscle samples from pigs that were fed with 2%-SWA exhibited the highest % of total lipids (5.26% DM) with no significant differences with the other groups. It would be important to confirm these results with a carcass with similar intramuscular content since it is known that there is a high correlation between intramuscular fat and meat lipid composition [46]. Ruqqia et al. [47] tested 13 seaweed extract from different species for hypolipidaemic potential in normal rats, and they found that some of these extracts caused a decrease in total serum cholesterol, triglyceride, and LDL cholesterol but an increase in

HDL cholesterol. These results sugges<sup>t</sup> that not all seaweed has the same effect on cholesterol metabolism. Since seaweed represents a group of organisms with diverse types of bioactive compounds, further studies are needed to understand the biological effects of the extract of *Macrocystis pyrifera* on cholesterol metabolism.

The proximal composition of the SWA used in this study is described in Table 1. Lipid content, crude protein, and ash content are lower than the values reported by Ortiz et al. [14] in the fresh seaweed (*Macrocystis piryfera*). These authors reported (in DM) 10.8% for ash, 13.2% for crude protein and 0.7% for total lipids, and 75.5% for carbohydrates. Seaweed composition depends on the harvest conditions, the habitat, and many other external conditions such as water temperature, light intensity, and nutrient concentration in the water [48].

The fatty acid composition of intramuscular tissue is affected by dietary lipid composition, de novo lipogenesis, desaturation, and the difference in the utilization of various fatty acids by the animal body [49]. In LL samples from this study, palmitic acid (C16:0) was the most abundant SFA, oleic acid (C18:1*c n* −9) was the most abundant MUFA, and linoleic acid (C18:2 *<sup>n</sup>*−6) the most abundant PUFA in the LL of the examined pigs. The composition of the fatty acids of the LL samples from this study is similar to those reported by Alonso et al. [50] and Parunovic et al. [51] in pork meat with no dietary seaweed inclusion. Comparing the fatty acids composition of LL samples with *Macrocystis pyrifera* seaweeds, the most abundant MUFA was also 18:1*c n* −9 (oleic acid) with 19.64 ± 0.08 %; the linoleic acid (18:2 *<sup>n</sup>*−6) reached values of 43.41% and the predominant SFA was also palmitic acid (C16:0; 16.17 ± 0.06) [14]. El Bahr et al. [52] reported a significant increase in the levels of EPA, DHA, total PUFA, and arachidonic acid in breast muscle of broiler chickens fed with microalgae extracts (1 g/kg diet), suggesting that high contents of methionine and lysine (present in the microalgae) were positively correlated with the increase in PUFA. *Macrocystis pyrifera* contains low contents of proteins and essential amino acids like methionine and lysine [14] compared to a microalgae, like *Arthrospira* sp. [27].

Several studies found that the long-chain ω-3 PUFA content in the muscle or adipose tissue was largely independent on the timing of feeding ω-3-PUFA-rich diets [53]; in this study, pigs were fed with the SWA for 45 days during the fattening phase; however, the effect of dietary SWA was not significant. Perhaps, it is necessary to incorporate the SWA during the growing and/or finishing phase to evaluate the impact on meat chemical composition.

Fat and fatty acids are important because of their effects on human health. In this study, the index value H/h and IA ratios turned out to be less than 1 (Supplementary Table S1), which indicated that regardless of the inclusion of SWA in the diets, all samples are categorized as healthy meats. The relation between H/h comes from the functional effects of fatty acids in cholesterol metabolism and gives a superior measure of the nutritional evaluation of fats from a nutritional standpoint [35]. The relation H/h is a suitable indicator to evaluate the risk of elevated blood cholesterol since it excludes C18:0 but includes two important hypercholesterolemic, palmitic acid (C14:0) and oleic acid (C16:0) are known to be the most important hypercholesterolemic fatty acids [35].

The balance in the relationship ω6 and ω3 ( ω6/ ω3) plays an important prevention role for severe chronic disorders and autoimmune diseases, and these authors agree to the average recommended value of 5:1. The ω6/ ω3 ratio, which is currently recommended by the OMS, should be lower than 10, in *M. piryfera* this ratio is 7.42 [54].

The inclusion of dietary SWA affected the content of ash; however, in this study, microminerals such as Mn, Fe, and Cu were found in less quantity in meat samples from animals that consumed the greatest proportion of SWA (4%). All seaweeds are characterized by a higher ash content (19.3–27.8% DM) than those observed in edible plants [21], being considered by some authors [27] as an important organic source of minerals for livestock nutrition; however, there is wide variability in mineral content among seaweed species. *Macrosysty pyrifera*, for example, is rich in Mg (39 ± 2.8 mg/g), Na (36.9 ± 9.9 mg/g), K (67.5 ± 22.3 mg/g) and Fe (117 mg/kg) [11,14]. On the other hand, these algae have a relatively low content of Mn (11 mg/kg), Zn (12 mg/kg), and Cu (2 mg/kg) compared

to other species of brown seaweed [11,21]. There is no scientific evidence of the direct relationship between the mineral content of the dietary seaweed additive and the mineral content in meat. The numerous bioactive compounds that are present in fed additive could affect the chemical composition of meat. Moreover, the content of polysaccharides such as alginates and agar or carrageenan could cause the formation of insoluble complexes with minerals, decreasing their bioavailability [55].

It has also been reported that *Laminaria* spp. is rich in alginates, which probably hampers the bioavailability of Ca, and that the apparent absorption values of Na and K were significantly higher in rats supplemented with *Laminaria* spp. while Mg absorption was not affected [56]. Several components in fed matrices can also exhibit retention properties in minerals, such as phenolic compounds and phytic acid, which reduce the bioavailability of Fe and Zn.
