The higher IMF content of Iberian pigs reared outdoors compared to those reared in intensive conditions and fed on concentrated diets is well known by farmers and dry-cured ham producers [44] and was previously reported [13,23]. This could be due to the high intake during the fattening period prior to slaughter of acorns which have a high caloric value (Rodríguez-Estévez et al. [20] estimated a daily feed intake of 2.92 kg DM acorn) and low protein content with an unbalanced amino-acid profile [8]. In the current study, there were no differences in IMF between FR pigs and LP pigs, but there were differences between both previous and SP pigs in SV. These results could be explained by the low protein content of the LP diet, which was similar to that of FR, and lower than that for the SP treatment. When insufficient dietary protein content is provided to pigs, excess energy is diverted to fat deposition [6]. Moreover, evidence suggests that, with low-protein diets, lipogenic enzymes are expressed more readily in muscle than in subcutaneous fat [45]. Therefore, diet composition, particularly the protein/energy ratio, can be used to increase fatness, with a consequent effect on performance [46]. Indeed, feeding pigs ad libitum with protein- or lysine-deficient but adequate-energy diets during the growing or finishing phases was shown to increase IMF proportion [36], which corroborates our results. In this sense, Schiavon et al. [47] found higher fat cover and thickness and marbling in hams from heavy pigs fed low-protein diets. More recently, Li et al. [38] evidenced the effects of low-protein diets on variations in the expression of two genes (*ACC* (acetyl-CoA carboxylase alpha) and *HSL* (hormone-sensitive lipase)) related to lipid metabolism, thereby promoting fat deposition in the muscle, which agrees with the results of the present study. The effect of feeding diets on chemical meat composition was only observed in SV. IMF content was lower in LL than in SV, which could be related to the type of muscle metabolism, as it is generally accepted that a higher proportion of oxidative fibers implies a greater IMF content [44].

With respect to color, the higher luminosity (L\*) values in SV from pigs reared in the FR system than in that from pigs reared in intensive conditions (LP and SP) could be attributed to the combined effect of feed characteristics and the environment, and not only to the exercise of pigs, as demonstrated by López-Bote et al. [48] in studies with Iberian pigs. Thus, the higher L\* value of SV from FR compared to LP and SP pigs could also be related to the higher IMF content in FR animals, in accordance with the results of Andrés et al. [44], who found a positive relationship between L\* value and fat content in Iberian pork. In the same way, Tejerina et al. [14] found that pigs reared in extensive conditions with acorns and grass had higher L\* and b\* values in LL and SV than those from Iberian pigs raised in intensive conditions with concentrated diets. In our study, LL showed a same tendency as SV, albeit without significant differences, which could be related to the lower IMF content in LL than in SV, in accordance with Tejerina et al. [14].

#### *4.4. Fatty-Acid Composition*

It is well known that the fatty-acid composition of pig tissues is affected principally by the fatty-acid composition of feed [49]. In our study, the influence of feeding background on the fatty-acid composition of porcine tissues was reflected more markedly in subcutaneous fat than in LL and SV muscles. Even though pigs fed in confinement (SP and LP diets) had monounsaturated fatty-acid-enriched diets, a significant influence of FR on the four major fatty acids (C18:1 n-9, C16:0, C18:0, and C18:2 n-6) and SFA and PUFA of subcutaneous fat was found. FR pigs showed a higher C18:1 n-9 content at the level of the tailbone than SP and LP, reflecting the high concentration of oleic acid from acorns, in agreement with the results previously reported [50]. However, Ventanas et al. [13], studying the effect of extensive feeding vs oleic acid-enriched mixed diets in Iberian pigs, did not detect any effect on C18:1 n-9. Moreover, in our study, FR pigs exhibited a lower SFA and higher PUFA content than LP and SP, in accordance with previous studies comparing Iberian pigs fed on free-range and concentrated diets [23]. Nevertheless, the level of protein in the diet did not affect the fatty-acid composition of subcutaneous fat and LL. The greatest effect of the level of protein in diets was reflected in SV, with the biggest change being in C18:2 n-6, and subsequently total PUFA, with the LP regime exhibiting lower proportions than both FR and SP regimes. These results are in agreement with Wood et al. [6], who reported that C18:2 n-6 and C18:1 n-9 are the fatty acids whose concentrations are most affected by a reduction of protein in pig diets. Previous works showed that these two fatty acids are those most affected by changes in total fat deposition in pigs and other animal species [51].  The linoleic acid obtained from the diet is then

progressively diluted as fat synthesis increases, which explains the declining concentration of this fatty acid. Nevertheless, no significant effect of protein level in the diet was detected for C18:1 n-9 content in our study. The explanation of this aspect could be based on the fatty-acid composition of concentrated feed used in our study, which was rich in oleic acid. Hence, dietary protein content (and, more specifically, the protein/energy ratio) may be used to modify the degree of carcass fat and fatty-acid composition. It is generally accepted that the high sensory quality of Iberian pig meat products from pigs fed under extensive conditions when compared to those fed on mixed feeds is attributed to variation in the content and fatty-acid composition of intramuscular lipids [52,53].

#### **5. Conclusions**

The results of this trial show that low-protein diets during the final fattening period prior to slaughter, similar to a feeding regime in free-range conditions with acorns and grass, do not affect Iberian pig productive traits. Additionally, they increase IMF content, which is one of the quality parameters most appreciated by consumers. In this sense, there is growing interest in muscle tissue recently, owing to the effect of increasing IMF on meat quality. Thus, feeding Iberian pigs with LP diets should be an interesting strategy to improve the quality of Iberian pig meat and dry-cured products.

**Supplementary Materials:** Estimation of the intake of acorns and grass by Iberian pigs in extensive rearing system. The following are available online at http://www.mdpi.com/2076-2615/10/2/273/s1.

**Author Contributions:** Conceptualization, J.F.T. and E.G.; methodology, J.F.T., E.G., M.P., and A.H.-M.; data analysis, E.G.; animal management, E.G. and M.P.; writing—original draft preparation, J.F.T., E.G., and A.H.-M.; writing—review, J.F.T., E.G., A.H.-M., and M.P.; writing—editing, J.F.T.; project administration and funding acquisition, J.F.T. and E.G. All authors read and agreed to the published version of the manuscript.

**Funding:** The research was supported by the regional government of Extremadura and the European Social Fund (Research Project PRI08B091).

**Acknowledgments:** The authors gratefully acknowledge the workers at the Valdesequera farm (CICYTEX) for their technical assistance in animal management.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Meat Quality, Amino Acid, and Fatty Acid Composition of Liangshan Pigs at Di**ff**erent Weights**

**Mailin Gan 1,2,**†**, Linyuan Shen 1,2,**†**, Lei Chen 1,2, Dongmei Jiang 1,2, Yanzhi Jiang 3, Qiang Li 4, Ying Chen 4, Guihua Ge 4, Yihui Liu 4, Xu Xu 4, Xuewei Li 1,2, Shunhua Zhang 1,2,\* and Li Zhu 1,2,\***

Received: 27 March 2020; Accepted: 5 May 2020; Published: 9 May 2020

**Simple Summary:** The research on the quality of traditional pork can not only provide a reference for the thorough breeding and food development of pigs, but also make a reference for understanding the local history and social culture.  It is mainly raised in the Liangshan Yi area and is closely related to the dietary culture of the local people. The characteristics of, and changes in, the meat quality, amino acid composition and fatty acid composition of Liangshan pigs of different weights were revealed for the first time in this paper. It was found that as the weight of Liangshan pigs increased, the contents of marbling score, intramuscular fat, shear force, Met, Asp, Asn, C18: 0 and C20: 2 increased, and drip loss, Trp and C22: 6 decreased. Taken together, our findings serve as a reference for the development of the local Liangshan pig industry.

**Abstract:** Indigenous pig breeds are important biological resources and their diversity has been severely damaged.  Here, the meat quality, amino acid, and fatty acid composition of Liangshan pigs were compared at seven stages within the weight range of 50–90 kg. A score for comprehensive factors of meat quality was maintained after rising and kept in a plateau within 74.9–91.5 kg of body weight. The total amount of amino acids in the longissimus dorsi muscle remained stable, and the total fatty acids showed an upward trend. Amino acid composition analysis revealed that as the body weight of Liangshan pigs increased, umami, basic, and acidic amino acid contents decreased, while the essential amino acids (EAA) content and the ratio of basic amino acids to acidic amino acids increased. Fatty acid composition analysis revealed that as body weight increased, the content of polyunsaturated fatty acids (PUFA) exhibited a downward trend, while the content of saturated fatty acids (SFA) exhibited an upward trend. This study is a primary step towards the development and utilization of Liangshan pigs and provides useful information for local pork processing and genetic improvement.

**Keywords:** Liangshan pig; meat quality; amino acid; fatty acid; traditional pig products

#### **1. Introduction**

Local pigs are important biological resources for new breeds and strains, the protection of animal diversity, and the realization of sustainable animal husbandry [1]. Pig production and breeding have rapidly entered globalization alongside economic globalization. Duroc, Yorkshire, Landrace, and Buckshire pigs represent the majority of breeds on the market, whereas many local pig breeds are endangered [2]. It is worth noting that pig breeding has long pursued high growth rates and high lean meat rates, which has led to a decline in pork quality, such as meat color, shear force and flavor [3]. However, consumers have recently begun to pursue pork of a higher quality and richer flavor. Therefore, local pig breeds are a resource that could meet the diverse needs of consumers [4].

The formation of local pig breeds is closely related to the local environment and people's consuming habits [5]. Pigs are an important part of local society and culture. In-depth studies of pork quality not only provide a reference for improved breeding and food development but can also provide insights into local history and social culture. The development and utilization of local pig breeds is an important way to protect local pig resources and diet culture and is of great significance for local economic development and national cultural heritage.

 It has a strong resistance to cold and thrives on coarse feed. Like most local pigs, Liangshan pigs have strong adaptability and good meat quality. However, Liangshan pigs have a slow growth rate and low feed conversion rate; therefore, the population of Liangshan pigs has decreased rapidly in recent years [6]. Limited information exists on the Liangshan pig breed; therefore, the goal was to acquire basic information of different quality characteristics to be used a future reference in the development and utilization.

In the present study, the quality, and amino acid and fatty acid composition of meat from 140 slaughtered Liangshan pigs was measured. The analysis of these data will help towards understanding meat quality characteristics and change rules of Liangshan pigs, and to formulate optimal slaughter times and suitable food development strategies. The results of this study are also of reference value for the genetic improvement of other local pigs and the development of specialty foods.

#### **2. Materials and Methods**

The experimental protocol was approved by the Animal Care and Ethics Committee of Sichuan Agricultural University, Sichuan, China, under permit No. DKY-S20123030 and No. DKY-S20123138.

#### *2.1. Animals*

The experiment was organized and performed at the Liangshan pig conservation farm of Mabian Gold Liangshan Agriculture Development Co. Ltd. (Sichuan, China). A total of 140 pigs with a similar birth date and birth weight (half barrows and half gilts) were randomly selected from the farm. Based on the methods of previous reports, 140 pigs were slaughtered at 7 different weight stages (the difference between each stage was approximately 6 kg) between 160 and 260 days of age, with 20 pigs from each stage (Table 1). The ingredients of the basal experiment diets are shown in Table S1.

**Table 1.** Information of Liangshan pigs being slaughtered.

#### *2.2. Management*

All pigs were fed the same commercial feedstuff. The pigs had ad libitum access to diet and water. All pigs were slaughtered following the method of Xiao et al. [7]. After transport to the abattoir, the pigs had no access to feed for 24 h before slaughter.

#### *2.3. Meat Quality Trait Measurements*

The determination of meat quality traits mainly refers to those used in our previous study [1]. The longissimus dorsi muscle samples used to measure meat quality traits were collected from the left side of the carcass adjacent to the last rib, within 45 min after slaughter. The penultimate 3–4 intercostal samples (the thickness is about 3 cm) of the longissimus dorsi muscle were used to measure pH, color and marbling scores (MS), and the samples (about 300–500 g) of the last rib of the longissimus dorsi muscle was used to measure drip loss, cooking loss and shear force (SF). The meat samples' pH was determined using a pH meter (model 720A; Orion Research Inc., Boston, MA, USA) according to the procedure of Alonso et al. [8]. The first measurement was to measure the central 1/3 location area of the meat sample at 45 min post-mortem (pH1), and the second at 24 h (pH2). Make 3 repetitions for each sample, take 3 readings for each repetition, and then calculate the average. Color parameters were measured using a Minolta CR-300 colorimeter (Minolta Camera, Osaka, Japan). Drip loss was calculated from the weight loss of a sample (approximately 30 g) wrapped in foil and placed on a flat plastic grid after storage for 24 h at 4 ◦C. Cooking loss was determined by cooking meat samples for 30 min, then, after cooling, measuring the weight loss relative to the uncooked weight. Marbling scores (MS) were determined using longissimus dorsi muscle 24 h after slaughter (colorimetric method, 5-point scale; the larger the score value, the richer the muscle fat content). Shear force (SF) was determined using a Texture Analyzer (TA.XT. Plus, Stable Micro Systems, Godalming, UK) equipped with a Warner-Bratzler shearing device.

#### *2.4. Analysis of Free Amino Acids and Fatty Acids*

Free amino acid (FAA) and fatty acid compositions were determined according to a previous article [1]. FAA composition was measured using liquid chromatography–mass spectrometry (Liquid phase: LC-20AD, Shimadzu, Japan; Mass Spectrometry: 5500 Q TRAP LC-MS/MS, AB SCIEX, Framingham, MA, USA), and gas chromatography–mass spectrometry (GC-MS 7890B-5977A, Agilent, Palo Alto, CA, USA) was used to detect fatty acid composition.

#### *2.5. Meat Chemical Composition*

Intramuscular fat (IMF), crude protein (CP) and ash contents were measured by the Nutrition Institute of Sichuan Agricultural University. CP was determined by the Kjeldahl method, and IMF content was determined by Soxhlet extraction [9].

#### *2.6. Statistical Analyses*

The ANOVA procedure was performed in SAS for Windows Release 8.0 (SAS Institute Inc., Cary, NC, USA) and was used to analyze the data collected. Duncan's test was used for comparing the mean values of the results. Mean values and standard errors are shown in the tables, with differences considered significant if *p* < 0.05. A comprehensive evaluation of Liangshan pigs at different bodyweight stages was performed using a factor analysis test.

#### **3. Results**

#### *3.1. Meat Quality and Meat Crude Chemical Composition*

The meat quality and crude chemical composition of Liangshan pig meat samples exhibited significant differences at different stages (Table 2). The first stage exhibited the highest L\*2 and drip loss values, while the marbling score, shear force, crude protein, and intramuscular fat content were the lowest. At the seventh stage, meat samples' pH1, shear force, and intramuscular fat content were the highest, while drip loss and ash value were the lowest.

**Table 2.** Meat quality of Liangshan pig at different stages.

pH1 and L\*1 measured at 45 min postmortem; pH2 and L\*2 measured at 24 h postmortem. S.E. standard error, a, b, c and \* mean significant difference.

With the increase in slaughter weight, meat samples' pH1, pH2, cooking loss, and crude protein increased slowly and fluctuated, while marbling score, shear force and intramuscular fat content rapidly and continuously increased (Figure 1A,B). As the slaughter weight increased, L\*1, L\*2 and ash decreased slowly and fluctuated, while drip loss rapidly and continuously decreased (Figure 1C,D). The overall analysis score for of Liangshan pig quality factors first increased and then remained at high levels with further increases in bodyweight (Figure 1E,F).

**Figure 1.** Change pattern of Liangshan pig meat quality traits. (**A**) Slowly increasing meat quality traits. (**B**) Rapidly increasing meat quality traits. (**C**) Slowly falling meat quality traits. (**D**) Rapidly falling meat quality traits. (**E**) Crushed stone graph for factor analysis of meat quality traits. (**F**) Comprehensive score for factor analysis of meat quality traits.

#### *3.2. Free Amino Acid Contents*

It can be seen in Table 3 and Figure 2A that the total amino acid (TAA) content in the longissimus dorsi muscle of Liangshan pigs slightly fluctuated (<30%) at different stages; the highest value was in the first stage and the lowest was in the second stage. Lys, Ile, Val, Trp, His, Arg, Glu, Tyr and Ala contents in Liangshan pig longissimus dorsi muscle were highest in the first stage, and Thr, Gln, Gly and Pro were the highest in the third stage (Table 3). Leu, Met, Ser and Asp contents were highest in the sixth stage, and Ile and Asn were the highest in the seventh stage.

The content of essential amino acids (EAA) in the longissimus dorsi muscle of Liangshan pigs in the seventh stage was the highest, reaching 28.13% (Figure 2A). Further analysis revealed that as the slaughter weight increased, the composition of basic amino acids and acidic amino acids in the longissimus dorsi muscle showed a downward trend and fluctuated, while the ratio of basic amino acids to acidic amino acids increased in fluctuation (Figure 2B). Sweet and umami amino acids were highest in the fifth stage, while bitter amino acids were highest in the sixth stage (Figure 2C).

#### *3.3. Fatty Acid Levels*

A total of 24 fatty acids were measured in the longissimus dorsi muscle of Liangshan pigs in the seven stages tested (Table 4). C16:0, C18:1, C18:2, C18:0, C20:4 and C14:0 are contained in more than 1%, and the cumulative proportion of these fatty acids exceeded 96% in the seven stages (Figure 3A). The C18:1 content was highest in the fourth stage, and the C16:0 content was highest in all stages other than stage six. The saturated fatty acids (SFA) was the lowest in the first stage and the highest in the fifth stage. Monounsaturated fatty acid (MUFA) content was highest in the fourth stage and lowest in the seventh stage. Polyunsaturated fatty acids (PUFA) content was highest in the first stage and lowest in the fifth stage (Figure 3B,C). Further analysis revealed that the overall n6 and n3 content showed a downward trend, while the n6:n3 values increased in volatility (Figure 3D).

**Table 3.** The amino acid content of longissimus dorsi muscle of Liangshan pig (mg/100g).

a, b, c and \* mean significant difference.

**Figure 2.** Analysis of amino acid composition in the longissimus dorsi muscle. (**A**) Changes in total amino acid (TAA) content and changes in essential amino acids (EAA) and non-essential amino acids (NEAA). (**B**) The content of basic and acidic amino acids. (**C**) The amino acid ratios of longissimus dorsi muscle with different flavors. Umami AA: Glu, Asp; Sweet AA: Gly, Ala, Ser, Thr, Pro, Gln, Lys; Bitter AA: Tyr, Arg, His, Val, Met, Ile, Leu, Trp, Phe.

**Table 4.** Fatty acid content of the longissimus dorsi muscle of the Liangshan pig (mg/100g).

a, b, c and \* mean significant difference.

**Figure 3.** Analysis of fatty acid composition and characteristics in the longissimus dorsi muscle. (**A**) The content of the top 6 fatty acids. (**B**) A composition of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in longissimus dorsi muscle. (**C**) Changes in SFA, MUFA and PUFA contents. (**D**) The ratio of n6:n3 of longissimus dorsi muscle.

#### *3.4. Comprehensive Meat Quality Evaluation of Liangshan Pigs at Di*ff*erent Bodyweight Stages*

Correlation analysis was performed on meat quality indicators that changed by>50% of their values at the first stage. Mar bling score was significantly positively correlated with shear force, intramuscular fat, and C18:0 content. Shear force was significantly positively correlated with intramuscular fat and C18:0 content. Intramuscular fat content was significantly positively correlated with Asp and C18:0 content, and significantly negatively correlated with C22:6 content (Table 5).

**Table 5.** Correlation analysis of the indexes of longissimus dorsi in Liangshan pig.

\* mean significant difference.

Through factor analysis, three characteristic values greater than 1 were obtained, and the cumulative contribution rate of the components reached 91.18% (Figure 4A). The component matrix results are shown in Table 6. The first principal component (PC) is mainly related to mar bling score, shear force and C18:0 content, the second principal component is mainly related to C22:6 content, and the third principal component is mainly related to C20:2 content (Table 6). As can be seen from Figure 4B, the comprehensive score shows a trend of first increasing, then decreasing (Figure 4B).

**Figure 4.** Results of the factor analysis. (**A**) A crushed stone graph. (**B**) A comprehensive score for factor analysis.

**Table 6.** Rotated component matrix.

PC: principal component. 1: Only the principal components with feature values greater than 1 are displayed.

#### **4.  Like most of the world's local pig breeds, Liangshan pigs are endangered [10,11]. The natural environment of the Liangshan Yi area and the dietary culture of the local people have determined the characteristics of Liangshan pigs. However, little is known about the basic biological characteristics of the Liangshan pig. Many studies have shown that age and weight are the most important factors affecting meat quality [12,13]. Here, meat quality traits, and amino acid and fatty acid composition of the longissimus dorsi muscle of Liangshan pigs were measured at seven stages (between 50 and 90 kg bodyweight) and were analyzed for their characteristics and changes.

Studies on the development of animal tissues and organs have shown that fat deposition occurs later than muscle deposition, and fat deposition is rapid after the turning point in animal growth [14]. Our previous results showed that the Liangshan pig growth turning point was 193.4 days at 62.5 kg [6]. In the present study, mar bling score and IMF content increased rapidly with weight gain. As Liangshan pigs' weight increased, the shear force of the longissimus dorsi muscle rapidly increased, which may be due to the gradual growth of the muscle fi ber diameter and an increase in muscle connective tissue content [15]. Drip loss decreased as slaughter weight increased. The effect of weight on drip loss is consistent with the findings of other reports [13,16].

Amino acids are basic units that make up proteins required by animals [17]. EAAs must be o btained directly from food, which is extremely important for maintaining the body's nitrogen balance and health [18]. The total amount of amino acids in the longissimus dorsi muscle of Liangshan pigs at different weights remained relatively stable, but EAA content showed an upward trend. Amino acid composition is also related to the taste of meat. Amino acids are normally divided into sweet amino acids, bitter amino acids, and umami amino acids [19,20]. The sweet and bitter amino acid contents of Liangshan pigs were relatively sta ble at different bodyweights, but umami amino acid content gradually decreased. Approximately 30% of umami amino acids were lost by the seventh stage compared to the first stage.

Amino acids are also divided into neutral amino acids, basic amino acids, and acidic amino acids [21]. The acidity and basicity of amino acids are usually determined according to the num ber of car boxyl groups and amino groups. Amino acids with more car boxyl groups than amino groups per molecule are termed 'acidic' (Asp and Glu) [22], otherwise they are termed 'basic' (Arg, Lys and His) [23]. Interestingly, as the weight of Liangshan pigs increased in this study, basic and acidic amino acid contents decreased, while the ratio of basic to acidic amino acids increased. This may be a reason for the increase in Liangshan pig meat sample pH as weight increased.

As bodyweight increased, the total fatty acid content of Liangshan pig meat samples showed an upward trend, which was consistent with intramuscular fat content and mar bling score. Dietary fatty acids are closely related to cardiovascular health, and higher SFA content in meat products has been shown to affect cholesterol metabolism [24]. PUFAs possess many physiological functions [25], such as maintaining biofilm structures, treating cardiovascular diseases [26], anti-inflammation [27], and the promotion of brain development [28]. It is worth noting that as the weight of Liangshan pigs increased, SFA content showed an upward trend, while the changes of PUFA were symmetrical with SFA. Further analysis revealed that n6:n3 values in Liangshan pig meat samples showed a rising trend of volatility.

Among the 54 indicators measured in this study, 10 indicators changed by more than 50%. Among these indicators, three were of meat quality traits, four were amino acids, and three were fatty acids. Correlation analysis showed that as intramuscular fat was deposited, C18:0 and Asp content increased rapidly, while C22:6 content decreased rapidly. Although saturated fatty acids are associated with a higher risk of cardiovascular disease, C18:0 does not lead to an increase in blood cholesterol [24]. Asp is an umami amino acid, and an increase in Asp can improve the taste of pork [29]. Further factor analysis shows that, in the fourth to seventh stages, the comprehensive score was higher, which is consistent with the factor analysis results based on meat quality traits. A similar pattern was also found in other pig breeds [16].

#### **5. Conclusions**

The current results show that differences in meat quality, amino acid composition, and fatty acid composition are present in Liangshan pigs at different slaughter weights. As bodyweight increased, mar bling score, intramuscular fat, shear force, Met, Asp, Asn, C18:0, and C20:2 content increased, and drip loss, Trp and C22:6 content decreased. The comprehensive factor score first increased and then decrease with weight gain within 74.9–91.5 kg of bodyweight. When slaughtering between 74.9 and 80.4 kg, the meat quality of Liangshan pigs is the best. Slaughtering between 74.5 and 80.4 kg provides the best meat quality in Liangshan pigs. In addition, when slaughtered at 80.4 kg, pork had the highest sweet amino acid content and the lowest n6:n3 ratio. Therefore, considering the meat quality, amino acid composition and fatty acid composition, the suitable slaughter weight of Liangshan pigs is 74.9–80.4 kg. This study provides effective data for the genetic improvement and specialty food processing of Liangshan pigs and provides new insights and references for research into local high incidences of disease.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/10/5/822/s1, Table S1: Ingredients of the basal experiment diets.

**Author Contributions:** Conceptualization: M.G., L.Z. and X.L.; Data curation: L.S. and L.C.; Funding acquisition: D.J., Q.L. and Y.J.; Methodology: Y.C. and G.G.; Project administration: L.C.; Supervision: X.X. and Y.L.; Validation: M.G. and S.Z.; Writing—original draft: M.G.; Writing—review & editing: L.S. and L.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (No. 31972524; No. 31530073), the Sichuan Science and Technology Support Program (No. 2016NYZ0050; No. SCCXTD-009SCSZTD-3-008), the earmarked fund for China Agriculture Research System (No. CARS-36-05 B).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

1. Gan, M.; Shen, L.; Fan, Y.; Guo, Z.; Liu, B.; Chen, L.; Tang, G.; Jiang, Y.; Li, X.; Zhang, S.; et al. High altitude adaptability and meat quality in tibetan pigs: A reference for local pork processing and genetic improvement. *Animals* **2019**, *9*, 1080. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **Portuguese Local Pig Breeds: Genotype E**ff**ects on Meat and Fat Quality Traits**

**José Manuel Martins 1,\* , Rita Fialho 2, André Albuquerque 2 , José Neves 1, Amadeu Freitas 1, José Tirapicos Nunes 3 and Rui Charneca 3**

Received: 19 March 2020; Accepted: 19 May 2020; Published: 22 May 2020

**Simple Summary:** Local breeds are generally associated with slower growth rates, higher slaughter weights, and fatter carcasses due to genetic and rearing system characteristics. When compared to intensive pig production systems, those based on European local breeds generally provide a more favourable response to the required increase in the production of high-quality pork and pork products in sustainable chains, meeting consumer demands. Reducing costs and improving the economic viability of production systems while preserving the quality of the products obtained is of vital importance. In this work, we propose that Portuguese local pig production chains could improve their performance and productivity without compromising the quality of the final product by crossing local breeds instead of crossing with modern breeds. This could help to maintain or increase local breed populations, supporting conservation of animal biodiversity.

**Abstract:** This work investigated the contribution of cross-breeding between two local Portuguese pig breeds to the conservation of animal biodiversity and income of local pig producers.  Pigs were reared outdoors, fed ad libitum, and slaughtered at ~65 (trial 1) and 150 kg BW (trial 2). In trial 1, AL pigs showed higher SM intramuscular fat, lower total collagen, and higher soluble collagen than BI pigs, while AL × BI and BI × AL pigs showed intermediate (NS) values. AL, AL × BI, and BI × AL pigs showed higher SM myoglobin content, and AL a more intense red colour than BI pigs. Finally, AL, AL × BI, and BI × AL showed higher total lipids in DSF than BI pigs. In trial 2, SM and DSF results were similar to those obtained in trial 1. In GM, AL and BI × AL showed higher intramuscular fat than BI and AL × BI pigs, while AL, AL × BI and BI × AL showed lower total collagen content than BI pigs. In conclusion, these results suggest that RI crosses are a productive alternative, with overall muscle and DSF traits statistically not different between AL × BI and BI × AL, and similar to those observed in AL pigs.

**Keywords:** swine; Alentejano; Bísaro; Ribatejano; meat quality; dorsal subcutaneous fat

#### **1. Introduction**

The increasing demand for pork products is leading to an international effort to save traditional pig breeds and develop new breeds [1]. In Portugal, the main local pig breeds are the Alentejano (AL), an Iberian type breed [2] with an extreme genetic closeness to the Iberian pig [3], and the Bísaro (BI) pig, from the Celtic type [2]. The AL pig is characterised by a low growth rate (except under "montanheira" regime) and precociously high adipogenic activity [4].  These environmentally well-adapted local breeds are less productive than modern improved genotypes, and their production chains depend mainly on the marketing of meat, and fermented and dry-cured meat products with highly valued sensory characteristics [5–7]. Almost extinct in the 1980s [5,8], these two breeds and their traditional systems have recovered since the 1990s [6], driven by consumer concerns about animal welfare, sustainable production, and meat and meat product quality issues. Although still threatened (AL) and rare (BI) breeds [9], they currently have a high ecological, economic and social importance in their production regions [6,10,11]. Increased yields and reduced costs on these productive systems is a continuous challenge, and crossbreeding is one way of achieving it.

AL and BI breeds homelands, in the South and North of Portugal respectively [8], have contact zones in the Tagus River region. In these contact zones (e.g., Ribatejo region), crosses between the two breeds were common until the 1950s [12] and the meat and meat products obtained were highly appreciated. However, there are no data available regarding these crosses, popularly called Ribatejano (RI) pigs. This study, included in the TREASURE project dedicated to European local pig breeds, was the first to collect and analyse carcass, pork and fat data from crossbred RI pigs, using AL and BI pure animals as controls. It represents a potential new management strategy for these breeds' production chains, while attending to societal demands for environment preservation, sustainable local agro-economy, as well as to consumers demands for quality and healthiness on regional pork products. The recovery and commercial use of these crosses could also help to preserve the pure breed populations, maintaining animal biodiversity, essential for an efficient and sustainable world food production and to meet the different needs of modern human societies [13]. In fact, loss of biodiversity may lead to an impaired response to changing environments.

Following a previous work where growth, carcass traits and loin data were presented [14], this study evaluates meat and fat quality from AL, BI, and AL × BI and BI × AL (RI crosses) pigs, reared outdoors, fed ad libitum, and slaughtered at ~65 and 150 kg BW.

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Design*

Experimental procedures and animal care were performed in compliance with the ethical guidelines and regulations of the Portuguese Animal Nutrition and Welfare Commission (DGAV—Directorate-General for Food and Veterinary, Portugal), following the 2010/63/EU Directive.

Composed by two sequential trials, this work had four experimental groups, with pure Alentejano (AL) and Bísaro (BI) pigs and their reciprocal crosses, AL × BI and BI × AL (Ribatejano (RI) pigs, the common name of these crossbred). Male pigs (n = 20 for each of the 4 genotypes) surgically castrated within the 1st week of age were reared outdoors from 28.6 ± 0.5 kg BW (mean ± SEM) until ~65 kg (trial 1) and from 65.2 ± 0.4 kg to ~150 kg (trial 2). In trial 1, pigs were group-fed with commercial diets (15.5–16.6 g/100 g crude protein, 12.4–12.7 g/100 g NDF, 4.5–5 g/100 g total lipids and 14.1–14.3 MJ/kg DE—Supplementary Table S1) at estimated ad libitum consumption [15], in a single daily meal (09:00 h). In trial 2, pigs were individually fed with commercial diets (15.4–16.6 g/100 g crude protein, 12.4–12.9 g/100 g NDF, 4.7–5 g/100 g total lipids and 14.1–14.3 MJ/kg DE—Table S1) and diet refusals were measured daily. All animals had free access to water and were weighed every fortnight.

Temperature and relative humidity data were registered throughout the experimental work. Average temperature, average minimal and maximal temperature, and average relative humidity were, respectively, 11.2, 6.5, 17.4 ◦C, and 75.0% in trial 1 (January–April), and 21.9, 13.7, 31.1 ◦C, and 56.3% in trial 2 (April–October).

Slaughtered at a commercial slaughterhouse in three batches per slaughter weight, animals had free access to water but were fasted for ~16 h during lairage. Ten animals from each genotype were slaughtered at the end of trial 1 (average BW of 64.2 ± 0.3 kg) and nine animals at the end of trial 2 (average of 150.6 ± 0.9 kg BW) by exsanguination following CO2 stunning. Commercially reared local pigs are slaughtered at lighter weights for consumption as fresh meat or roasted pigs, or at heavier weights for the production of high quality traditional fermented and cured products [6,16] with PDO or PGI European certification.

#### *2.2. Muscle and Adipose Tissue Sampling*

Samples of SM muscle and of DSF were obtained from the left half carcasses at ~65 and 150 kg BW, while samples of GM muscle were collected only at ~150 kg. Samples were vacuum packaged and frozen (−20 ◦C) until analysis.

#### *2.3. Muscles and Adipose Tissue Analyses*

Leg (SM and GM) muscles represent a cut of greatest economic value and mass. Muscles ultimate pH (pHu) was determined 24 h post-mortem by a pH-meter with a puncture electrode (LoT406-M6-DXK-S7/25, Mettler-Toledo GmbH, Germany).

Moisture was determined in muscles and DSF according to ISO-1442 [17]. Total nitrogen from muscle samples was determined in a Leco FP-528 (Leco Corp., St. Joseph, MI, USA) by the Dumas combustion method (method 992.15) and from DSF samples by the Kjeldahl method (method 928.08) [18]. Total protein content was estimated as total nitrogen × 6.25. Total lipids were determined in muscles according to Folch, et al. [19] and in DSF by Soxhlet extraction (method 991.36) [18]. Muscles myoglobin and total collagen were determined as previously described [20] and soluble collagen according to Hill [21].

Surface colour measurements [22] of raw SM and GM samples were recorded with a CR-400 colorimeter (Konica Minolta Sensing Europe B.V., Nieuwegein, Netherlands) with a D65 illuminant, after blooming for 30 min. Individual CIE (Commission Internationale de l'Éclairage) *L*\* (lightness), *a*\* (redness) and *b*\* (yellowness) values were averaged out of six random readings across muscle surface. The same procedure (without blooming) was applied to DSF samples. Chroma (C∗ = √- *a*∗2 + *b*∗2 , hue angle - H = *tan*−1(*b*∗ /*a*∗) , and saturation (*C*∗ /*L*∗) were calculated.

#### *2.4. Statistical Analysis*

All data were tested for normality by the Shapiro-Wilk test. Results are presented as mean ± rSD. Data were analysed by one-way analysis of variance (ANOVA) with SPSS Statistics software (IBM SPSS Statistics for Windows, v24.0, IBM Corp., Armonk, NY, USA). Mean differences were considered statistically significant when *p* < 0.05, and *p* values between 0.05 and 0.10 were considered trends.

#### **3. Results**

#### *3.1. Trial 1: Pigs Slaughtered at ~65 kg BW*

Pigs were slaughtered at an average age of 186.2 ± 2.9 days.

#### 3.1.1. Muscle Tissue Analyses

SM physico-chemical data were affected by genotype (Table 1). Moisture was lower (*p* < 0.05) in AL and AL × BI than in BI pigs, while total intramuscular fat (IMF) was higher (*p* < 0.05) in AL than in BI pigs. Myoglobin content was higher (*p* < 0.01) in AL pigs and Ribatejano (RI) crosses than in BI pigs, whereas total collagen was lower (*p* < 0.05) in AL than in BI pigs, with their crosses showing intermediate values. Regarding soluble collagen, as a % of total collagen, values were higher (*p* < 0.05) in SM from AL than BI pigs. SM pHu values were not affected by genotype, but significant differences were observed in colour parameters (Table 1). Lightness (*L*\*) was lower and redness (*a*\*) higher (*p* < 0.05) on AL than BI pigs, again with their crosses showing intermediate values. These results affected hue angle (H◦) and saturation, respectively lower (*p* < 0.01) and higher (*p* < 0.05) in AL than BI pigs (Table 1).

**Table 1.** Chemical composition, pH, and CIE colour values of *Semimembranosus* muscle from Alentejano (AL), Bísaro (BI), AL × BI and BI × AL pigs slaughtered at ~65 kg BW (*n* = 10 for each genotype).

CIE—Commission Internationale de l'Éclairage. AL × BI and BI × AL represent the reciprocal crosses of the commonly known Ribatejano pig. DM—Dry matter. a,b,c Values in the same row with different superscript letters are significantly different (*p* < 0.05).

#### 3.1.2. Adipose Tissue Analyses

Chemical composition of DSF was also affected by genotype (Table 2). Moisture content was lower (*p* < 0.001) in AL than in BI pigs, with RI crosses showing intermediate values. Total lipids, inversely related to moisture content, were higher (*p* < 0.001) in AL and RI crosses than in BI pigs (Table 2).

**Table 2.** Chemical composition, and CIE colour values of dorsal subcutaneous fat from Alentejano (AL), Bísaro (BI), AL × BI and BI × AL pigs slaughtered at ~65 kg BW (*n* = 10 for each genotype).

AL × BI and BI × AL represent the reciprocal crosses of the commonly known Ribatejano pig. a,b,c Values in the same row with different superscript letters are significantly different (*p* < 0.05).

#### *3.2. Trial 2: Pigs Slaughtered at ~150 kg BW*

Pigs were slaughtered at an average age of 353.6 ± 2.6 days.

#### 3.2.1. Muscle Tissues Analyses

Physicochemical data from SM samples were less affected by genotype in the fattening period. IMF was higher in AL and BI × AL than in BI pigs, but this difference did not attain statistical significance (Table 3). Total collagen was lower (*p* < 0.05) in AL pigs and RI crosses than in BI pigs, with soluble collagen (% total collagen) following the opposite trend without attaining statistical difference. SM pHu was higher (*p* < 0.05) in AL than in BI pigs (Table 3). Regarding colour, genotype only tended to affect lightness (*L*\*) (*p* = 0.067) and yellowness (*b*\*) (*p* = 0.059) values, and therefore hue angle (H◦) (*p* < 0.05) was lower in AL and AL × BI than in BI pigs (Table 3).

**Table 3.** Chemical composition, pH, and CIE colour values of *Semimembranosus* muscle from Alentejano (AL), Bísaro (BI), AL × BI and BI × AL pigs slaughtered at ~150 kg BW (*n* = 9 for each genotype).

AL × BI and BI × AL represent the reciprocal crosses of the commonly known Ribatejano pig. a,b Values in the same row with different superscript letters are significantly different (*p* < 0.05).

IMF from GM muscle was higher (*p* < 0.01) in AL and BI × AL than in BI and AL × BI pigs, while total collagen was lower (*p* < 0.01) in AL pigs and RI crosses than in BI pigs. However, soluble collagen (% total collagen) and colour parameters of GM were not affected by genotype (Table 4).

AL × BI and BI × AL represent the reciprocal crosses of the commonly known Ribatejano pig. a,b Values in the same row with different superscript letters are significantly different (*p* < 0.05).

#### 3.2.2. Adipose Tissue Analyses

Chemical data from DSF were affected by genotype, with total protein lower (*p* < 0.001) in AL pigs and RI crosses than in BI pigs, and total lipids higher (*p* < 0.05) in AL than in BI and AL × BI pigs (Table 5). Finally, DSF colour parameters were also influenced by genotype. Redness (*a*\*), chroma (C\*), and saturation were lower (*p* < 0.05) in AL than in BI pigs, while hue angle (H◦) was higher (*p* = 0.05), with RI crosses showing intermediate values.

**Table 5.** Chemical composition and CIE colour values of dorsal subcutaneous fat from Alentejano (AL), Bísaro (BI), AL × BI and BI × AL pigs slaughtered at ~150 kg BW (*n* = 9 for each genotype).

AL × BI and BI × AL represent the reciprocal crosses of the commonly known Ribatejano pig. a,b,c Values in the same row with different superscript letters are significantly different (*p* < 0.05).

#### **4. Discussion**

Sustainability and animal welfare policies are increasingly being adopted by the food industry in response to consumer demands. These changes can help strengthen pork niche markets and broaden the target audience for small farmers practicing outdoor swine production [23]. However, farmers and researchers must find a way to improve productivity and product quality, and scientifically support product differentiation [23,24]. One way to improve the performance of outdoor finishing pigs is through crossbreeding. AL and (mainly) BI genotypes are not sufficiently studied, namely in the case of muscles other than longissimus lumborum. In addition, currently available information was obtained in trials with very different or not even described rearing and feeding conditions, as well as age/slaughter weights, among other aspects. Therefore, additional studies are required to evaluate different production stages, in controlled experimental environments.

In order to evaluate meat and fat quality from AL and BI pigs, as well as their reciprocal crosses reared outdoors and fed ad libitum with commercial diets, animals were slaughtered at ~65 and 150 kg BW.

#### *4.1. Trial 1: Pigs Slaughtered at ~65 kg BW*

Growth and carcass data from this trial were previously presented and discussed by Martins et al. [14]. Briefly, AL pigs had a shorter carcass length and lower bone cuts weight than BI, while Ribatejano (RI) crosses showed intermediate values. AL pigs also showed low lean and high fat cuts proportions, while in BI pigs lean cut proportions were more important. This agrees with the presence in both genotypes of the LEPR c.1987T allele, usually associated with higher fatness, that is almost fixed in the fatty AL when compared to the leaner BI pig breed (0.98 vs. 0.26 frequencies, respectively) [3]. The lower lean and higher fat cuts of the carcasses from AL pigs led to a 12.1% lower commercial yield and 44.7% higher fat cuts proportion than those observed for BI. These differences were due to changes in untrimmed shoulder (−9.7%), loin (−15.8%) and untrimmed ham (−13.7%), and in belly (+24.4%) and backfat (+89.5%) cuts. Meanwhile, although RI crosses showed overall intermediate values, their fat cuts proportions were not significantly different from those of BI pigs. These differences led to

lower lean-to-fat cuts ratio and higher backfat thickness and ZP ("Zwei punkte") fat depth in AL than in BI pigs [14].

Muscle physicochemical traits were affected by genotype. In SM, IMF content was 25.5% higher in AL than in BI pigs, confirming the precociously high adipogenic activity in the AL pig [4]. Strongly influenced by genotype, IMF is positively correlated with the sensory properties, juiciness and palatability of meat [25–27]. Meanwhile, compared to IMF values observed in 100 kg AL pigs fed at 85% ad libitum [20,28], and BI pigs [29], those observed in our trial were slightly higher in AL and similar in BI. The differences in the AL breed results, were probably due to different feeding conditions used in both trials.

Myoglobin content has been suggested as a genotype-related characteristic [30]. In our trial, myoglobin showed higher values in the SM muscle from AL pigs and RI crosses when compared to BI pigs. On the other hand, SM total and soluble collagen were respectively 17.4% lower and 25% higher in AL than BI pigs, with RI crosses showing intermediate values. Collagen proteins are the predominant constituents of skeletal muscle connective tissue network and a contributing factor to meat's texture [27,31]. Likewise, IMF content affects muscle cut resistance, with higher fat corresponding to lower shear force values [27,32]. Therefore, differences observed in IMF and collagen content of SM suggest a more tender meat in growing AL pigs, and tenderness is described as the most important factor for the perceived sensory quality of pork [33]. This trend was also observed in the longissimus lumborum (LL) samples from these animals [14]. Still, total collagen values were higher in SM than in LL, confirming that hindquarter muscles used for locomotion such as biceps femoris, semimembranosus, and semitendinosus, are inherently tougher than support muscles such as longissimus lumborum [27]. Finally, the pHu values, not affected by genotype, were close to the lower value of the normal range in pork, which varies between 5.5 and 5.8 [34].

Consumer's critical first impression of meat depends mainly on colour, which is in turn largely associated to myoglobin concentration and its chemical form. Other factors, such as the physical state of meat, including pH value, protein state, denaturation degree, and water loss, are also important [30]. SM values for colour coordinates in the literature are scarce and vary widely among breeds (e.g., 36–57, 3–17, and 4–15, for *L*\*, *a*\*, and *b*\*, respectively) [7,20,29,35–37]. Values observed in this trial are within the above-mentioned ranges. In our trial, the SM lowest levels of *L*\* and H◦ and the highest levels of *a*\* and saturation were observed in AL pigs, indicating a darker and redder meat [27]. This agrees with the previously mentioned higher myoglobin content in this genotype. Thus, when compared to BI, AL pigs showed a more intense red SM muscle, as observed in LL [14], which is a distinctive feature for the consumer [38]. The higher *L*\* values observed in SM samples from BI pigs could be partially associated to a higher muscle water loss in these pigs, already noticed in LL muscle [14]. In fact, the higher amount of muscle free-water provides a more reflective surface for light and is positively correlated to lightness [27,39]. Finally, colour values detected in muscle samples from RI crosses were overall intermediate to those observed in AL and BI genotypes, except for H◦, closer to the AL values in BI × AL and to the BI values in AL × BI pigs. The lower H◦ values observed in BI × AL pigs measure a colour closer to the true red axis and agree with a significantly high content in myoglobin in these pigs. Similar results were previously observed in biceps femoris from Duroc × Iberian pigs, when compared to Iberian × Duroc [40].

DSF chemical composition was also affected by genotype, with AL pigs showing a 33.6% lower moisture and an 18.9% higher total lipids content than BI pigs, confirming precociously high adipogenic activity of AL [4].

#### *4.2. Trial 2: Pigs Slaughtered at ~150 kg BW*

Growth and carcass data from this trial were presented and discussed by Martins et al. [14]. Briefly, in the fattening period AL pigs had a lighter bone structure and a more compact body than BI, presenting a shorter carcass, and lower bone cuts proportions. Carcass yields, higher in these older and heavier pigs, increased 1.16, 0.80, 1.17, and 0.97 percentage units for each 10 kg increase in BW

from 65 to 150 kg in AL, BI, AL × BI, and BI × AL pigs, respectively. This confirms fat deposition as the main responsible for increasing carcass yield in older pigs [41,42].

SM muscle was only affected by genotype in total collagen content and pHu parameters. IMF was 11% higher in AL than in BI pigs, however this difference did not attain statistical difference. When compared to BI, SM muscle samples from AL pigs also showed a higher percentage of IMF at 65 than at 150 kg BW (+23.4 and +11.1%, respectively). This was also observed in LL [14], suggesting that AL is an early maturing breed. Total protein values from AL were comparable to those previously reported for 100 kg castrated AL pigs fed at 85% ad libitum [20,28], but IMF values were higher, probably due to different feeding regimes and slaughter weight. Total protein and IMF values from BI pigs from our trial were identical to the ones reported by Carvalho [29].

Although the myoglobin content was 10.3% higher in SM from AL when compared to BI pigs, this difference did not attain statistical significance, contrary to what was observed in trial 1. Meanwhile, myoglobin content increased in all genotypes between 65 and 150 kg BW (186 and 354 days of age), showing that pork gained a more intense colour with age [27], as previously observed in Iberian pigs [43]. On the other hand, SM total collagen content observed in this trial in free-range AL pigs, was higher than the one in 100 kg confined AL pigs [20]. At 150 kg BW, SM total collagen and soluble collagen were also 25.1% lower and 20.6% higher in AL than in BI pigs, respectively. RI crosses also showed lower total collagen values than BI pigs. These differences suggest a higher tenderness of pork from AL pigs and RI crosses. Furthermore, ageing animals show a higher number of stable bonds between collagen molecules, with the corresponding decrease in its solubility [27,43,44], as observed in our pigs slaughtered at 65 and 150 kg. As animals age, meat becomes tougher, mainly due to an increase in the percentage of heat-insoluble collagen bonds [27]. This is a more important factor in local pig breeds than in industrial genotypes, because the former are slaughtered at physiologically older ages.

Ultimate pH (pHu) of meat influences water-holding capacity, colour, tenderness, flavour and shelf life of meat [45] and therefore, is a main quality determinant [45,46]. The pHu values observed in SM were within the normal range for pork [34], but were affected by genotype. As already observed in LL [14], pHu values from SM samples were higher in AL than in BI pigs. This suggests a higher muscle glycogen content in BI pigs, positively correlated to lower pH values [47]. Generally, leaner animals have higher percentages of fast-contracting glycogen-rich type IIb or white fibres [48], with a glycolytic metabolism and higher ATP-ase activity, leading to lower pHu values than those observed in slow-contracting oxidative type Ia or red fibres [46].

Meat colour, the major visual factor affecting meat quality [27], was influenced in a less expressive way by genotype in SM during fattening than during growth, as previously observed in LL [14]. Genotype only affected H◦, which was significantly lower in SM samples from AL and AL × BI pigs. SM muscle from AL and AL × BI pigs also tended to show lower levels of *L*\* than BI pigs, but *a*\* values were not significantly different. A lower *L*\* and H◦, as observed in AL and AL × BI pigs, is related to a darker and redder meat surface in terms of real colour perception [27]. Both darkness and redness can be enhanced at higher pH values, as observed in the SM muscle from AL pigs. In these conditions, reducing and oxygen-consuming enzymes are decreasing the percentage of myoglobin in the oxygenated form, and light scattering is minimized because hydrated muscle proteins are not releasing free water [39]. Overall, when comparing the two trials, an increase in age/weight led to a reduction of *L*\* and an increase of *a*\* values, generally associated to pork with a darker red colour [27]. This difference in *L*\* values from the SM muscle of AL and BI pigs was higher than two units in both trials, which could affect consumers preferences and influence the decision to purchase [49].

Chemical characteristics of GM muscle were slightly affected by genotype. IMF content was higher in AL and BI × AL pigs than in BI and AL × BI pigs, and was comparable to that previously observed in 103 kg BW Iberian pigs fed at 90% ad libitum [50]. Although the difference in IMF between AL and

BI was expected, due to the higher adipogenic activity of the former breed [4], IMF values observed in BI × AL, are interesting. In fact, when analysing the IMF values obtained in this trial, BI × AL pigs showed a fat content in both muscles numerically close to the one from AL pigs, suggesting a maternal effect. Since the technological quality of fresh meat and meat products is mainly determined by the lipid fraction, this higher IMF content in the two valuable ham muscles from BI × AL pigs, is very important. Similar results were obtained when comparing IMF content of biceps femoris from hams of Iberian pigs to those of Duroc boars × Iberian dams and of Iberian boars × Duroc dams [51]. Finally, GM total collagen observed in AL pigs and RI crosses was lower than the one in BI pigs. When associated to higher IMF values, as also observed in SM muscle (and in LL muscle—[14]), this suggests a higher tenderness of pork from AL pigs and RI crosses.

DSF chemical composition also varied among genotypes, with AL pigs showing a 29.5% lower total protein and a 6.2% higher total lipids content than BI. Such changes agree with the more adipogenic profile of AL when compared to the leaner BI pig [4,5]. However, histological studies are needed to clarify if the difference in protein content is related to collagen or fat deposition. The latter could be obtained either through an increased adipose cell number in BI pigs and/or by a cell hypertrophy in AL pigs (fewer cells per gram of subcutaneous tissue). When calculating the fat weight deposited in DSF ((DSF weight × % DSF lipids)/100) at 65 and 150 kg BW, the values were 2.88 and 6.31 kg, respectively. Once again, BI × AL pigs showed a total lipids content similar to that of AL, and higher than the one from BI and AL × BI pigs, which is important from a technological point of view. Finally, regarding DSF colour, AL pigs had a 33% lower *a*\* value than BI, which affected C\*, H◦, and saturation values. The higher lipid concentration in DSF of AL pigs may have contributed to the dilution of blood vessels in this tissue, leading to lower values of *a*\* and saturation, also observed in 65 kg pigs but without attaining statistical significance. In fact, haemoglobin, the major colour pigment in blood, can also affect tissue colour [27].

#### **5.  SM muscle from AL pigs showed higher IMF, redder colour, and lower total collagen, features that could positively influence the consumer from a visual and/or an eating quality point of view. Ribatejano (RI) reciprocal crosses (AL × BI and BI × AL) showed overall intermediate features between AL and BI genotypes, but higher lean and lower fat cut proportions and backfat thickness than AL. On the other hand, they showed a SM muscle with a myoglobin content and colour characteristics in line with those observed in AL pigs. This suggests a redder and darker meat than the one from BI pigs, at a slaughter weight generally used for meat production. These features were overall similar in both muscles of pigs slaughtered at the end of the fattening period (~150 kg BW). At this slaughter weight, muscles from RI crosses also had a lower total collagen content, suggesting a darker, redder, and more tender meat for fresh and cured products than the one from BI pigs. Therefore, RI crosses have the potential to be sustainably reared outdoors and to produce high quality meat and fermented or dry-cured products. AL × BI, more easily reared in the north of Portugal (BI dams' homeland), could improve the meat and meat products quality when compared to the ones obtained with pure BI pigs. As to BI × AL, more easily reared in the south (AL dams' homeland), this cross has better commercial yield and primal cuts proportions, when compared to those obtained from pure AL pigs, without compromising meat and meat products quality. Finally, the production of high quality/certified products to attain better market prices can lead producers to increase animal and productivity numbers and therefore contribute to maintaining or increasing animal biodiversity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/10/5/905/s1, Table S1. Chemical composition (g/100 g) of the commercial diets fed to Alentejano (AL), Bísaro (BI), AL × BI and BI × AL pigs slaughtered at ~65 and 150 kg BW.

**Author Contributions:** Conceptualization, J.M.M., R.C. and J.T.N.; Methodology, J.M.M., R.F., A.A., J.N. and R.C.; Animal management, R.C., A.F., J.M.M. and J.N.; Data Analysis, J.M.M.; Writing—original draft preparation, J.M.M.; Writing—review, J.M.M., R.F., A.A., J.N., A.F., J.T.N. and R.C.; Writing—editing, J.M.M.; project administration and funding acquisition, R.C. and J.M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was conducted within the project TREASURE, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634476. The content of this paper reflects only the author's view and the European Union Agency is not responsible for any use that may be made of the information it contains. This research was also funded by Portuguese national funds through FCT—Foundation for Science and Technology under Project UIDB/05183/2020, and a research grant SFRH/BD/132215/2017 to A. Albuquerque.

**Conflicts of Interest:** The authors declare no conflict of interest. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **A Newly Identified LncRNA LncIMF4 Controls Adipogenesis of Porcine Intramuscular Preadipocyte through Attenuating Autophagy to Inhibit Lipolysis**

#### **Yunmei Sun** †**, Rui Cai** †**, Yingqian Wang, Rui Zhao, Jin Qin and Weijun Pang \***

Laboratory of Animal Fat Deposition and Muscle Development, Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China<EMAIL_ADDRESS>(Y.S<EMAIL_ADDRESS>(R.C<EMAIL_ADDRESS>(Y.W<EMAIL_ADDRESS>(R.Z<EMAIL_ADDRESS>(J.Q.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-029-87091893

† These authors contributed equally to this work.

Received: 9 April 2020; Accepted: 18 May 2020; Published: 26 May 2020

**Simple Summary:** Compared with lean-type pigs, the intramuscular fat content of fat-type Bamei pigs was greater. LncRNA, as a vital regular, plays an important role in numerous biological processes. However, there were a few studies on the role of lncRNAs during IMF development in pigs. Based on these, lncRNA sequencing in intramuscular adipocytes was performed to explore the effects of lncRNA on intramuscular fat deposition. RNA sequencing analysis of intramuscular adipocyte from Bamei pig (fat-type) and Yorkshire pig (lean-type) indicated that, a novel lncRNA, lncIMF4, was associated with intramuscular adipogenesis. In addition, further researches showed that knockdown lncIMF4 promoted proliferation and adipogenic differentiation of porcine intramuscular adipocytes, whereas inhibited autophagy. Moreover, knockdown lncIMF4 facilitated intramuscular adipogenesis through attenuating autophagy to repress the lipolysis. Our findings will contribute to better understand the mechanism of lncRNA controlling adipogenesis in pig. Furthermore, it also provides a new perspective to study the role of lncRNA in regulating porcine intramuscular adipogenesis for promoting pork quality.

**Abstract:** Intramuscular fat (IMF) is implicated in juiciness, tenderness, and flavor of pork.  LncRNA is a vital regulator that contributes to adipogenesis. However, it is unknown about the regulation of lncRNA on IMF content. Here, by RNA sequence analysis of intramuscular adipocyte from Bamei pig (fat-type) and Yorkshire pig (lean-type), we found that a novel lncRNA, lncIMF4, was associated with adipogenesis. LncIMF4, abundant in adipose, differently expressed along with intramuscular preadipocyte proliferation and differentiation. Meanwhile, it is located both in cytoplasm and nucleus. Besides, lncIMF4 knockdown promoted proliferation and differentiation of porcine intramuscular preadipocytes, whereas inhibited autophagy. Moreover, lncIMF4 knockdown facilitated intramuscular adipogenesis through attenuating autophagy to repress the lipolysis. Our findings will contribute to understand better the mechanism of lncRNA controlling intramuscular adipogenesis for promoting pork quality.

**Keywords:** pork quality; lncIMF4; intramuscular preadipocyte; differentiation; autophagy

#### **1. Introduction**

Adipose tissue is essential for animals. It plays a vital role in the composition of living organisms and various metabolic processes. Among them, intramuscular adipose relates to meat quality.

Moderate intramuscular fat content can increase the tenderness and flavor of pork. The content of intramuscular fat (IMF) and its fatty acid composition play an important role in meat quality, affecting the sensory properties (juiciness, flavor, and tenderness), and nutritional value of meat [1]. The positive effects of IMF related to the quality of meat have been confirmed in pork [2], mutton [3], and beef [4]. The content depends not only on the amount of precursor fat cells converted to mature IMF cells but also on the deposition of lipid droplets in the IMF cells and lipid droplets in the myocytes [5].

LncRNA, a long noncoding RNA, participates in multiple life processes. They participated in many cellular biological processes, such as proliferation, differentiation, and apoptosis, by regulating the expression of their target genes [6–8]. There is some evidence demonstrated that lncRNAs regulated adipogenesis through multiple mechanisms. Hundreds of lncRNAs were involved in the regulatory network of adipogenesis [9,10]. More and more researchers are paying attention to study the influence of lncRNA on pig fat deposition and exploring its regulation for increasing meat quality. At present, many studies about lncRNA sequencing in adipose tissue from different pig breeds were performed. These sequencing analyses illustrated that many lncRNAs were involved in the development of porcine adipose tissue [11–13]. However, there were a few studies on the role of lncRNAs during IMF development in pigs. This exciting area needs to be further researched.

Autophagy is a process of phagocytizing its cytoplasmic protein or organelle and coating it into vesicles and fusing with lysosomes to form autophagosomes. Autophagy degrade the contents by autophagosomes to regulate cell metabolism. In addition, it updates to specific organelles; cell autophagy, like apoptosis and cell senescence, is an important biological phenomenon involved in the development and growth of organisms. Macroautophagy, is the best understood among the known autophagic pathways. It is characterized by forming a double-membrane structure called the autophagosome that fuses with the lysosome [14]. The study found that there was also the occurrence of macroautophagy in adipocytes. The lysosome contains various hydrolases; therefore, autophagy can degrade multiple cytoplasmic components and provide the resultant molecular building blocks, such as amino acids, glucose, nucleotides, and fatty acids [15]. Experiments in mouse models have shown that autophagy is required to maintain the levels of amino acids and glucose in blood and tissues of neonatal and adult mice during fasting [16–18]. Therefore, in order to investigate the mechanism of lncRNA on regulating adipose development, studies about the relationship between lipogenesis and autophagy require further exploration.

In our previous studies, we performed lncRNA sequencing in preadipocytes of porcine longissimus dorsi muscle during differentiation [19]. The data showed that lncIMF4 is a novel lncRNA. It was differentially expressed in different time groups. This inspired that lncIMF4 may be a potential target for adipogenesis. Additionally, bioinformatics analysis predicted that lncIMF4 participated in many biological processes including cell proliferation, differentiation, and autophagy. Therefore, this research on the function of lncIMF4 in intramuscular adipocytes was performed to better understand the mechanism of lncRNA controlling intramuscular adipogenesis for promoting pork quality.

#### **2. Materials and Methods**

#### *2.1. Animal*

Animal samples used in this study were approved by the Animal Care and Use Committee of Northwest A&F University. To investigate the expression pattern of lncIMF4 in pig, tissue samples from heart, liver, spleen, lungs, kidney, fat, and skeletal muscle (longissimus dorsi) of Bamei pigs at 3 days old (n = 4) and 180 days old (n = 4) were collected from experimental farm of Northwest A&F University (Yangling, China). All tissues for expression profile were immediately frozen in liquid nitrogen and then were kept at −80 ◦C until RNA isolation.

#### *2.2. Cell Culture*

The experiments refer to in vitro intramuscular adipocytes from Bamei and Yorkshire pigs. Intramuscular preadipocytes were isolated from longissimus dorsi muscle (LD) of 3-day-old piglets as described previously [19]. In brief, we used 0.2% collagenase I (270 U/mg; Gibco, Carlsbad, CA, USA) to digest samples at 37 ◦C in the water bath shaker for 2 h. Then, samples were sequentially filtered through 70 and 200 mesh filters to separate the cells. After washing twice with DMEM/F12, cells were seeded in dishes containing DMEM/F12 medium with 10% fetal bovine serum (Gibco, Australia). After 1.5 h, we rinsed off unattached cells. When density of cells reaches to approximately 100%, we used the cocktail method to induce adipocytes differentiation. The four differentiation stages were observed when cells were cultured in differentiation medium for 0 d (undifferentiated), 2 d (early differentiated), 4 d (middle differentiated), and 8 d (last differentiated). The autophagy was induced by a serum-free medium for 3 h after 6-day differentiation. Until that, preadipocytes were cultured in DMEM/F12 to proliferate. Briefly, we used OPTI to dilute negative control (NC) (or lncIMF4 siRNA to a concentration of 50 nM/mL), kept still for 5 min, then added Roche transfection reagent, gently blew, and further kept for 20 min. This mixed OPTI was added to the new medium.

#### *2.3. Cell Transfection*

For research on proliferation, 50 nM siRNA or negative control (NC) were transfected into cells by X-tremeGENE siRNA Transfection Reagent (Roche, San Francisco, USA) and Opti-MEM (Gibco, Grand Island, USA) when the density was 40%. After transfection for 24 h, the cells were harvested. For differentiation, when the density reached 80%, siRNA or NC were transfected, and differentiation medium was replaced with growth medium when the cells reached fusion. The siRNA and NC were obtained from RiboBio (Guangzhou, China).

#### *2.4. Hematoxylin–Eosin Staining*

The longissimus dorsi muscle tissue was taken from the fifth to sixth lumbar vertebrae of the 180-day-old Bamei pigs (n = 6) and the Yorkshire pigs (n = 6) and placed in the fixative. Longissimus dorsi muscle (LM) tissues were dehydrated by alcohol. The dehydrated tissues were made into paraffin sections. For HE staining, the sections were deparaffinized with xylene for 10 min. Then, the sections were washed with the distilled water and then were first placed in an aqueous solution of hematoxylin for several minutes. Then the sections were immersed in acid water and ammonia water for color separation for several seconds, respectively. Next, the sections were rinsed under the running water for 1 h, and then were immersed into distilled water for a while. Slices were dehydrated in 70% and 90% alcohol for 10 min, respectively, and then were dyed in eosin staining solution for 2–3 min. The stained sections were dehydrated with pure ethanol, and the sections were made transparent by xylene. The transparent slices were mounted with Canada Balsam and covered with a coverslip. Sample images were captured using a Nikon TE2000 microscope (Nikon, Tokyo, Japan).

#### *2.5. Fluorescence in Situ Hybridization*

Cell slide was placed on the bottom of the 12-well plate; cells were washed for 5 min and then were fixed at room temperature for 10 min with 4% paraformaldehyde. Briefly, 200 uL prehybrid solution was added to each well and they were blocked at 37 ◦C for 30 min. For prehybridization, the hybridization solution was preheated at 37 ◦C. To protect it from the light, 2.5 μL 20 uM lncRNA FISH Probe Mix stock solution or internal reference FISH Probe Mix stock solution was added to 100 μL hybridization solution. The prehybridization solution in each well was discarded and 100 μL of the probe hybridization solution containing the probe was added; the light was avoided, and then it was hybridized overnight at 37 ◦C. DNA staining is protected from light. Further, staining was done with DAPI staining solution for 10 min, avoiding light, and the cells were washed 3 times for 5 min each

time. The cell slide was carefully removed from the well in dark and fixed on the loaded slide with a sealing tablet for fluorescence detection.

#### *2.6. Cytoplasmic and Nuclear RNA Extraction*

This method is referred to a previous research [20]. For the extraction of cytoplasmic and nuclear RNA fraction, intramuscular adipocytes were collected after 6-day differentiation. Cells were washed with PBS, suspended in lysis buffer (10 mM NaCl, 2 mM MgCl2, 10 mM pH 7.8 Tris-HCL, 5 mM DTT, and 0.5% Igepal CA 630), and then incubated on ice for 5 min. After centrifuging at 8000 rpm for 5 min, the supernatant was transferred to a new microcentrifuge tube subjected to cytoplasmic RNA extraction, while the pellet was resuspended with lysis buffer and subjected to nuclear RNA extraction. For the RNA extraction, the fractions were first incubated with Proteinase K (10 mg/mL) at 37 ◦C for 20 min and then mixed with TRIzol. RNA was separated by chloroform and precipitated by ethanol with 3 M sodium acetate (pH 5.2, 1/10 volume). The extracted RNA was dissolved into ddH2O and used for reversed transcribed and real-time PCR analysis.

#### *2.7. RNA Extraction and Real-Time PCR*

Total RNA was isolated by TRIzol reagent (Takara, Otsu, Japan). Afterwards, the reverse transcription of RNA was performed using kits (Takara, Otsu, Japan), following the manufacturer's instructions. Bio-Rad iQTM5 (Bio-Rad, Hercules, CA, USA) was used to perform RT-qPCR. Expression level of the listed genes and lncIMF4 were related to that of β-tubulin. The information of primers is shown in Table 1.

**Table 1.** Primers used for real-time quantitative PCR.

#### *2.8. Western Blotting*

The protocol of western blot was followed according to a previous study [10]. Briefly, adipocytes were split by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) by adding protease inhibitor (Pierce, WA, USA). The total protein sample was separated in the SDS-polyacrylamide gel. Then, it was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). Next, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 ◦C overnight followed by a secondary antibody at room temperature for 1.5 h. The antibodies Cyclin B, Cyclin D, Cyclin E, PPARγ, and AP2 were purchased from Santa Cruz (CA, USA); C/EBPα and SREBP-1 were purchased from Abcam; p62, LC, ATGL, and HSL were purchased from CST (Boston, MA, USA); and β-tubulin was purchased from Sungene Biotech (Shanghai, China).

#### *2.9. Cell Counting Kit (CCK8) and EdU Assays*

The protocol of cell counting (CCK8) and EdU assays were performed following a previous study [6]. Adipocytes were cultured in 96-well plates with 2.5 × 103 cells per well. Cells were treated with NC or siRNA. The proliferation rates were tested by the Cell Counting Kit 8 (CCK-8) following manufacturer's instructions. For EdU assay, cells were incubated with EdU for 2 h after transfecting them with NC or siRNA. Subsequently, sample images were captured using a Nikon TE2000 microscope (Nikon, Tokyo, Japan).

#### *2.10. Flow Cytometry*

Cells were seeded in 6-well plates with 4 × 105 cells per well. After 24 h, cells were transfected with NC or siRNA. Next, cells were washed three times with PBS and fixed with 70% alcohol overnight at −20 ◦C. Cells were then treated with 1 mg/mL RNase at 37 ◦C for 40 min before being stained with 50 mg/mL propidium iodide (PI) at 4 ◦C for 1 h. Lastly, samples were detected with FACSCalibur flow cytometry (Franklin Lakes, NJ, USA).

#### *2.11. Oil Red O Staining*

Oil Red O Staining was performed according to a previously published method [12]. After being fixed in 4% paraformaldehyde solution, cells were incubated with 0.5% Oil Red O for 30 min and washed three times with PBS; adipocytes were visualized by phase-contrast microscopy (Tokyo, Japan). Oil Red O dissolved in lipid droplets was extracted with 100% isopropanol and its relative concentrations were determined by measuring the absorbance at 510 nm.

#### *2.12. Monodansylcadaverine (MDC) Staining*

The cells were digested with trypsin and centrifuged, and the cell pellet was collected. Then the cells were washed once with wash buffer and the supernatant was discarded. Next, the cells were resuspended by adding the appropriate amount of wash buffer to adjust the cell density to 10. Precisely, 90 uL of cell suspension was taken into the EP tube and 10 uL of MDC solution was added and mixed gently. The solution was stained at room temperature for 30 min, and the cells were collected by centrifugation. Then, the cells were washed twice with wash buffer, 300 uL DAPI solution was added to it, and stood still at room temperature. After 5 min, the cells were washed 3 times with PBS. The cells were resuspended in the collection buffer, the resuspended droplets were added to the slide, and the coverslip was attached. The slide was observed under a fluorescence microscope, and cells were counted and photographed. Autophagy staining detection kit was purchased from Solarbio.

#### *2.13. Statistical Analysis*

The diagrams were created by GraphPad Prism 6.0. SEM represents the variation between sample means. Group differences were analyzed with Student's *t* test or one-way ANOVA using PASW Statistics 20 (SPSS, Chicago, IL, USA) (\*, *p* < 0.05; \*\*, *p* <0.01).

#### **3. Results**

#### *3.1. LncIMF4 May be A Novel LncRNA Implicated in Intramuscular Fat Deposition*

The meat quality of Bamei pig is better than Yorkshire pig probably highly influenced by the higher content of intramuscular adipocytes in Bamei pig. (Figure 1A,B). The lipid content in intramuscular adipocytes of Bamei pig was also more than Yorkshire (Figure 1C). Comparison of lncRNA sequences was performed between intramuscular adipocytes of Bamei pig and Yorkshire pig. The results showed that the level of lncIMF4 was higher in large white pig, and it presented rising first then falling pattern (Figure 1D). In addition, the alignment track showed that lncIMF4 is an unannotated lncRNA in the pig genome (Figure 1E). Interestingly, the result of Gene Ontology (GO) term analysis demonstrated

that it was related with lipolysis and autophagy (Figure 1F). Therefore, further research was carried out following these pathways.

**Figure 1.** LncIMF4 may be a novel lncRNA implicated in intramuscular adipogenesis. (**A**) The intramuscular fat (IMF) content of longissimus dorsi muscle (LM) between Bamei and Yorkshire were analyzed by Soxhlet extraction method. (**B**) Hematoxylin–eosin (HE) staining of LM between Bamei and Yorkshire. (**C**) Porcine intramuscular adipocytes at 0, 2, 4, and 8 d after inducing differentiation (cell density reached 100%). (**D**) Heatmap depicting long noncoding RNA (lncRNA) having at least 2-fold change in intramuscular adipocytes between fat-type and lean-type pig at four differentiation stages; black fragments denote lncIMF4. (**E**) The alignment track of lncIMF4. (**F**) GO term of lncIMF4. The results were representative of means ± SEM of three independent experiments.

#### *3.2. The Expression Pattern of LncIMF4 in Pig and Its Subcellular Location in Adipocyte*

Based on the above results, we supposed that lncIMF4 was related to IMF content. We detected the levels of lncIMF4 during intramuscular adipocytes proliferation and differentiation. The expression patterns both showed a trend of rising first and then decreasing, reaching a peak on the second day, which was consistent with the sequencing results (Figure 2A,B). Tissue expression results demonstrated that lncIMF4 is highly expressed in adipose tissue, not only in 3-day-old piglets (Figure 2C) but also in 180-day-old pig (Figure 2D). To confirm the localization of lncIMF4 in intramuscular adipocytes, we performed the fluorescence in situ hybridization, showing that lncIMF4 was localized in both nucleus and cytoplasm (Figure 2E). The relative expression levels of LncIMF4 in the nucleus and cytoplasm were consistent with it (Figure 2F).

**Figure 2.** The expression pattern of lncIMF4 in pig and its subcellular location in adipocyte. (**A**) The levels of lncIMF4 during porcine intramuscular adipocytes proliferation. (**B**) The levels of lncIMF4 during adipocytes differentiation.  (**E**) Subcellular localization of lncIMF4. (**F**) LncIMF4 expression in cytoplasm and nucleus. The results were representative of means ± SEM of three independent experiments.

#### *3.3. LncIMF4 Knockdown Promoted the Proliferation of Porcine Intramuscular Preadipocytes*

Proliferation is an important process for intramuscular adipose development. Therefore, we explored the function of lncIMF4 in the proliferation of intramuscular preadipocytes. We transfected

siRNA into proliferating intramuscular preadipocytes to knockdown lncIMF4. The efficiency of siRNA meets the requirements of subsequent experiments (Figure 3A). CCK8 assay showed that the cell number was increased (Figure 3B). Furthermore, by cell cycle analysis, we found that knockdown of lncIMF4 increased the percentage of S phase cells and decreased the percentage of G1 and G2 phage cells (Figure 3C,D). The result of EdU assay was consistent with it (Figure 3E,F). Meanwhile, knockdown of lncIMF4 increased the levels of cell cycle-related genes compared with that of NC (Figure 3G,H). These results indicated that knockdown of lncIMF4 promoted the intramuscular adipocytes proliferation.

**Figure 3.** Knockdown of lncIMF4 promoted intramuscular adipocytes proliferation. LncIMF4 negative control (NC) or siRNA were transfected into cells at 40% density. (**A**) The inhibition efficiency of lncIMF4 in porcine intramuscular adipocytes treated with lncIMF4 siRNA. (**B**) Cell count was measured by Cell Counting Kit 8 (CCK8). (**C**) Cell cycle analyses. (**D**) The statistics results of cell cycle analysis. (**E**) EdU assay was performed after transfection for 24 h. (**F**) The percentage of EdU positive cells/DAPI positive cells was quantified. (**G**) RT-qPCR analyzed the cell cycle genes, cyclin B, cyclin D, and cyclin E after transfection for 24 h. (**H**) Western blot of the cell cycle genes, cyclin B, cyclin D, and cyclin E, after transfection for 24 h. The results were representative of means ± SEM of three independent experiments. \*, *p* < 0.05; \*\*, *p* < 0.01.

#### *3.4. LncIMF4 Knockdown Promoted Intramuscular Adipogenic Di*ff*erentiation*

To explore the function of lncIMF4 during intramuscular adipocytes differentiation, we used the siRNA to knock down it, and the efficiency of siRNA reached the subsequence experiment requirement (Figure 4A). Oil Red O staining also showed a significant increase in intracellular lipid droplets in siRNA group (Figure 4B,C). Key genes of adipocytes differentiation were significantly increased after transfection with siRNA (Figure 4D). In addition, the protein levels of lipogenesis were upregulated (Figure 4E,F). Cumulatively, these results indicated that knockdown of lncIMF4 promoted adipocytes lipogenesis.

**Figure 4.** Knockdown of lncIMF4 promoted adipocytes lipogenesis. LncIMF4 siRNA or NC were transfected into cells at 70% density at 50 nM. (**A**) The inhibition efficiency of lncIMF4 in porcine intramuscular adipocytes after transfecting lncIMF4 siRNA with NC on 6 day of differentiation. (**B**) Oil Red O staining, scale bar = 100 μm. (**C**) Absorbance value at 510 nm after incubation with Oil Red O. (**D**) RT-qPCR analysis of adipogenic and lipolytic genes on 6 day of differentiation. (**E**) Western blot analysis of adipogenic genes on 6 day of differentiation. (**F**) The quantification of protein levels. The results were representative of means ± SEM of three independent experiments. \*, *p* < 0.05; \*\*, *p* < 0.01.

#### *3.5. LncIMF4 Knockdown Inhibited Lipolysis by Attenuating Autophagy in Porcine Intramuscular Adipocytes*

In this study, we constructed the autophagy model of porcine intramuscular adipocytes by using nonserum culture medium after 6 days. The results showed that the number of autophagy cell was increased (Figure 5A,B). The levels of ATG7 and Baclin1 were also upregulated (Figure 5C,D). These results indicated that the autophagy model was successfully constructed.

**Figure 5.** Autophagy model of porcine intramuscular adipocytes was constructed. (**A**) Monodansylcadaverine (MDC) staining of porcine intramuscular adipocytes after inducing autophagy by serum-free medium for 3 h. (**B**) The quantification of autophagic cells/normal cells. (**C**,**D**) RT-qPCR was carried out to detect the cell autophagic genes, ATG7 (**C**) and Beclin1 (**D**). Data were representative of means ± SEM of three independent experiments. \*, *p* < 0.05. NC, negative control; TM, treatment.

We predict that lncIMF4 is related to autophagy according to the results of GO term, which showed that lncIMF4 was enriched in autophagy-related pathway. Therefore, lncIMF4 were knockdown to test whether adipocytes autophagy was regulated. After transfection with siRNA, cells with green fluorescent dot particles were less than in the control group (Figure 6A,B). This indicated that autophagy was decreased when lncIMF4 was knocked down. The mRNA and protein levels of ATG7 and Baclin1 were consistent with it (Figure 6C). Meanwhile, the markers of autophagy, p62 were significantly upregulated and the LC3 were downregulated (Figure 6D). Furthermore, Oil Red O staining revealed that lncIMF4 knockdown promoted porcine intramuscular adipocyte differentiation in autophagy model (Figure 6E). Protein levels of PPARγ and AP2 showed the same result (Figure 6F). Consistently, the mRNA and protein levels of ATGL and HSL, the key genes of lipolysis, were both decreased after transfection with siRNA (Figure 6G,H). These results demonstrated that knockdown of lncIMF4 promoted porcine intramuscular adipocyte adipogenesis by attenuating autophagy. The function of lncIMF4 is shown in Figure 6I.

**Figure 6.** Knockdown of lncIMF4 inhibited lipolysis by downregulating autophagy. (**A**) MDC staining of porcine intramuscular adipocytes transfected into cells at 70% density at 50 nM after inducing autophagy by serum-free medium for 3 h. (**B**) The quantification of autophagic cells/normal cells. (**C**) RT-qPCR was carried out to detect the cell autophagic genes. (**D**) Western blot analysis of autophagic marker genes on 6 day of differentiation. (**E**) Oil Red O staining, scale bar = 100 μm. (**F**) Western blot analysis of adipogenic genes on 6 day of differentiation in autophagy model. (**G**) The mRNA expression level of lipolytic genes on 6 day of differentiation. (**H**) Western blot analysis and quantification of lipolytic protein levels. (**I**) The function of lncIMF4 in porcine intramuscular adipocytes. Data were representative of means ± SEM of three independent experiments. \*, *p* < 0.05; \*\*, *p* <0.01.

#### **4. Discussion**

Higher intramuscular fat content in pork plays an important role in improving meat quality [21]. Many factors are included in the biological processes. The regulatory role of lncRNA is an essential part

of it [22]. In our previous study, we performed the lncRNA sequencing and found some lncRNAs related to adipogenesis. lncIMF4 was a novel lncRNA, which is predicted to participate in cell differentiation and autophagy. These processes could affect fat content in adipocytes [23–25]. This indicated that lncIMF4 have an effect on lipid synthesis in porcine intramuscular adipocyte. Interestingly, further research demonstrated that lncIMF4 was located both in nucleus and cytoplasm, so the role of it may be complex. In this study, we used small interference to explore its function in adipocytes.

The development of adipose tissue includes adipocytes proliferation and differentiation [26]. Recently, studies reflected that lncRNAs affect cell proliferation through variety of signaling [27,28]. In this study, we found that the level of lncIMF4 changed regularly along with cell proliferation. Based on this, we suspected that lncIMF4 participated in the regulation of proliferation of porcine intramuscular adipocyte. Subsequent experimental results were consistent with this hypothesis. Further study showed that knockdown lncIMF4 promoted intramuscular adipocyte proliferation. It reflected that lncIMF4 was important for intramuscular adipose development. The adipocyte differentiation is an important process regulating lipid deposition in adipose tissue. Adipocytes hyperplasia arises in the early stage of differentiation. Then, small lipid droplets appear in the cells and merge into large droplets after many adipogenic factors are activated. Finally, adipocytes become hypertrophic. LncRNA has been known as a regulator to affect adipocyte differentiation [29–31]. However, there is little research on the role of lncRNA in porcine adipocyte differentiation. In our study, GO term analysis showed that lncIMF4 is associated with cell differentiation. Furthermore, the level of lncIMF4 is related to fat deposition closely. Therefore, we explored the effects of lncIMF4 on porcine intramuscular adipocyte. Consistently, the results demonstrated that knockdown lncIMF4 promoted adipogenic differentiation in porcine intramuscular adipocyte. It confirmed that lncIMF4 regulated porcine intramuscular adipocyte lipogenesis.

Autophagy is a biological process that maintains life survival by degrading organelles and proteins when there is lack of nutrition, energy, or imbalance of environment. Defective autophagy, is associated with numerous diseases, including neurological disorders, cancer, cardiomyopathies and metabolic disorders [32]. Autophagy has a dilution effect on high intracellular lipids and influences lipid metabolism in many ways, from lipogenesis to lipolysis [33,34]. The autophagosomes will wrap lipids to ablate if autophagy occurs in adipocytes. Autophagy and lipolysis are both decreased with enough food supply, while both are increased in nutrient deprivation [35]. In this research, bioinformatics analysis predicts that lncIMF4 may have a particular regulatory effect on autophagy. Autophagy can affect the differentiation and adipogenic processes of adipocytes. Many signaling pathways were involved in these processes [36]. Therefore, we hypothesized that lncIMF4 regulated lipolysis to affect lipid deposition through autophagy. This is a new insight to study the relationship between lncRNA and intramuscular adipose. The autophagy of normal cells is very weak. To explore this regulation, we firstly constructed an autophagy model of porcine intramuscular adipocyte. In recent years, the induction of autophagy has focused on the construction of nutrient-deficient environment and drug treatment. To reduce stress on cells and reduce cell loss, autophagy was induced by starvation treatment in this study. The appropriate induction time was determined to be 3 h after the addition of serum-free medium, which was more transient than the time in which the adipocytes induced autophagy in other literature. Further study showed that in autophagy model, knockdown lncIMF4 promoted lipogenesis and inhibited lipolysis. Moreover, it also attenuated autophagy in porcine intramuscular adipocyte. Generally, we confirmed that lncIMF4 knockdown promoted lipogenesis of porcine intramuscular preadipocyte through attenuating autophagy. The underlying mechanism will be the focus of our next experiment.

#### **5. Conclusions**

In conclusion, we identified lncIMF4 as a novel lncRNA, which is related to intramuscular fat deposition. lncIMF4, located in both nucleus and cytoplasm, knockdown promoted proliferation and differentiation of porcine intramuscular adipocytes, whereas inhibited lipolysis through attenuating autophagy. This research provides a new perspective to study the role of lncRNA in regulating porcine intramuscular adipogenesis for promoting pork quality.

**Author Contributions:** Conceptualization, W.P.; methodology, Y.S.; software, Y.W.; validation, R.Z.; investigation, R.C.; resources, W.P.; data curation, J.Q.; writing—original draft preparation, Y.S. and R.C.; writing—review and editing, Y.S. and R.C.; and funding acquisition, W.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by following funding: the National Natural Science Foundation (31872979 and 31572366), the National Key Research and Development Program of China (2017YFD0502002), and the Key Research and Development Program of Shaanxi Province (2018ZDXL-NY-02-03).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **A Short Period of Darkness after Mixing of Growing Pigs Intended for PDO Hams Production Reduces Skin Lesions**

#### **Lieta Marinelli 1, Paolo Mongillo 1,\* , Paolo Carnier 1 , Stefano Schiavon 2 and Luigi Gallo 2**

Received: 11 August 2020; Accepted: 19 September 2020; Published: 23 September 2020

**Simple Summary:** Mixing unacquainted growing pigs is a common practice in commercial herds to adjust the group size to the pen dimensions and to balance the body weights of pigs within pens. Aggressive behavior following regrouping may include fights that can result in skin lesions and detrimental economic effects. Strategies aimed at limiting such issues can therefore improve animal welfare in practice. In the present study, we investigated the effects of darkness, maintained for 48 h after the formation of new social groups, on the expression of agonistic behavior and on the accumulation of skin lesions of growing pigs. The provision of 48 h of darkness significantly reduced the number of skin lesions on the mid- and rear thirds of pigs' body. However, no corresponding reduction was observed in agonistic behavior, suggesting that darkness decreases the efficacy of aggressions, rather than how often or for how long they are expressed. Furthermore, an analysis of the location of lesions indicates that aggressions towards a fleeing companion, rather than reciprocal ones, were those mostly affected by darkness. The present results identify in the provision of darkness an easily applicable and relatively inexpensive intervention, that leads to the reduction of one of the most problematic consequences of agonistic interactions, i.e., skin lesions.

**Abstract:** Agonistic behavior after the regrouping of unfamiliar pigs has been recognized as one of the major welfare issues for pig husbandry, as it may result in lesions, lameness, and health problems. One scarcely investigated strategy to curb agonistic behavior is reducing the availability of visual stimuli potentially eliciting aggressions. In this study, we investigated the expression of agonistic behavior by growing pigs and the resulting accumulation of skin lesions over a period of 14 days following the formation of new social groups, which occurred in a condition of darkness maintained for 48 h. Compared to a simulated natural photoperiod (12 h light/day), darkness significantly reduced the number of skin lesions on the mid- and rear thirds of pigs' body (*p* ≤ 0.01). A lack of corresponding decrease in frequency and duration of agonistic interactions suggests that darkness acts by decreasing the efficacy, not the expression, of aggressions. Furthermore, the location of lesions mostly affected by darkness indicates that the latter mostly acted by reducing the possibility of pigs to convey damage to a fleeing conspecific, rather than to one involved in a reciprocal fighting. The lighting regime provided did not affect growth performance traits of a 17-weeks feeding trial. The present results identify in the provision of darkness an easily applicable, and relatively inexpensive intervention, that leads to the reduction of skin lesions.

**Keywords:** aggression; agonistic behavior; darkness; light; mixing; photoperiod; pig; regrouping; skin lesions; *Sus scrofa*

#### **1. Introduction**

Growing pigs in commercial herds are usually subjected to mixing events with unrelated and unacquainted animals [1]. This management procedure may occur several times from birth to slaughter [2] and generally aims at balancing the body weight of pigs within pens, in order to increase uniformity and to control stocking density by adjusting the group size to the pen dimensions [3]. Following regrouping, agonistic behavior may be intense, primarily serving to establish a new relative social ranking, thereby reducing future needs of disputes among animals [4]. Agonistic behavior can lead to fights, which result in skin lesions (SL), frequently used as a practical indicator of the extent and the severity of post-mixing aggressive behavior [5], as well as increased risk of lameness and of infections due to immunosuppressive effects [6]. Apart from the negative effects on animal welfare, the regrouping of unfamiliar pigs may also cause detrimental economic effects, negatively affecting growth rate and meat quality [3].

Agonistic behavior aimed at establishing relative social ranking in wild boars and feral pigs rarely ends with severe aggressions (i.e., bites), thanks to greater freedom of movement due to space allowance and fighting tactics based on complex and gradual behaviors [1,7]. Starting with reciprocal visual and olfactory inspection and gentle nudging, the interactions may escalate up to full-blown fights depending on signals indicating opponents' intention to submit [8–11]. When the overt fight is started, the behavioral patterns mainly aim at minimizing the risk of being bitten and increasing the likeliness of biting. Accordingly, strategies at regrouping should reduce the number of interactions that escalate to aggressions or reduce the efficacy of aggressions in terms of reciprocal injuries. In this respect, several studies have investigated the efficacy of manipulating housing system, pen dimension, stocking density, and group size as methods to reduce aggressive consequences [1,3]. The introduction of environmental enrichment, which may increase the opportunity to carry out exploratory behavior and diverting pigs' attention from conspecifics, has been explored [12], though with controversial results [1]. Other studies focused on the possibility to manipulate access to olfactory stimuli involved in the escalation of interactions by exposing animals to a diverse class of chemicals capable of altering olfactory information [13,14]. Much less attention has been paid to the possibility of manipulating visual signals and lighting regime; that has proven to affect growth rate and meat and ham quality of heavy pigs [15] and may be implicated in the escalation of inter-pig interactions.

To our knowledge, the manipulation of visual signals was only implemented by grouping animals after sunset [16,17], taking advantage of the lower phase of the circadian rhythm in a pig's activity. In these conditions, the number of aggressions was reduced by about 50% in the 90 min following mixing, but this effect disappeared the following morning, resulting in a comparable number of SL after three days from regrouping. Although these results suggest that darkness only delays aggressions, the beneficial effects of reducing visual information at mixing might have been biased due to the duration of the treatment used. Indeed, the higher amount of fighting between pigs occurs in the first 24–48 h after regrouping [6], which suggests a possible advantage in applying darkness for longer than one night. Moreover, if darkness affects aggression by reducing availability of opponents' visual information, extending its period of application could reduce outcome of aggressions (i.e., reciprocal injuries), regardless of their occurrence.

The aim of the present study was to deepen our knowledge on the effects of darkness at regrouping on aggressive behavior and skin lesions of growing pigs in a commercial farming situation.

#### **2. Materials and Methods**

#### *2.1. Animals, Housing, and Experimental Design*

The pigs involved in the present study are part of a larger feeding trial carried out at the research farm of the DAFNAE Department of the University of Padova, Italy [18–20]. Pigs were reared in accordance with EU Directive (2008/120/EC), and experimental procedures were reviewed and approved by the Ethical Committee for the Care and Use of Experimental Animals of the University of Padova, in accordance with the Italian legislation (D.Lgs. 26/2014, transposition of the EU 2010/63/UE directive).

Data were collected on 100 pigs (skin lesion counts) and 48 pigs (behavior observations)—specifically, barrows and gilts belonging to four different genetic types used in Italy for the production of heavy pigs to provide dry-cured hams. The piglets, who were born within the same week, arrived at the research farm around 80 d old and with a body weight (BW) close to 38 kg. At arrival, and until the beginning of the study, pigs were kept in four pens, homogeneous for genetic type (12 or 13 pigs/pen, two pens for each genetic type) and fed the same commercial diets according to the same feeding regime until the twenty-second week of age (86.5 ± 6.5 kg BW). Pens were located in two rooms (four pens/room), managed with the same ambient conditions and with an identical arrangement of pens within each room. Each pen was 5.8 × 3.8 m, had a slatted floor, and was equipped with a single-space electronic feeder (Compident Pig–MLP; Schauer Agrotronic, Prambachkirchen, Austria). Readers are referred to Schiavon et al. [18] for further details regarding the characteristics of pigs, their diets, growth performance, and carcass traits.

At the beginning of the study, the pigs were mixed, with the formation of new social groups, housed in the eight pens described above. Each of the eight groups was composed of 12 or 13 individuals (stocking density ≥ 1.6 m2/pig). Groups were balanced for BW, genetic types (e.g., each pen containing three or four pigs per genetic type), and sex (six or seven gilts and barrows per pen, and one or two of each sex, for each genetic type).

During mixing procedures (Day 0) and for the subsequent 48 h, a lighting regimen of darkness was kept in one of the two rooms (Figure 1), thus affecting half of the pigs (*n* = 50) in the study; darkness was obtained by covering the windows, the only source of ambient light in the room, with a fourfold black plastic polypropylene sheets. Simulated natural photoperiod (12 h light/day) was reintroduced in this room at h 10:00 of Day 2 and maintained for the rest of the experiment; that ended after two weeks at Day 14. In the other room, a lighting regimen of 12 h light/day was kept for the entire duration of the experiment (windows were covered in this room as well to obtain precise control on the photoperiod). Apart from the lighting regimen in the first 48 h following mixing procedures, pigs of the two rooms were managed and fed in the same way.

**Figure 1.** Schedule of skin lesion counting (on eight pens, *n* = 100 pigs) and behavior observations (on four pens, *n* = 48 pigs), relative to the day of mixing (Day 0); darkness was applied to half of the pens for the first 48 h after mixing (grey area).

#### *2.2. Skin Lesion Counts*

The total number of SL was recorded for each pig immediately before mixing (Day 0), after 72 h post-mixing (Day 3), 24 h after the natural photoperiod was reintroduced in the darkened room, and at Day 7 and Day 14 following regrouping (Figure 1). Lesions were recorded by direct observation of each pig, and it was performed independently by two trained observers who counted the total number of fresh lesions. A lesion was defined as a single and continuous scratch, regardless of severity, whereas freshness was judged on the basis of lesion color, usually red-pink, and development of

scabbing [21,22]. Lesions were recorded independently on three different locations of the body: front (head, neck, shoulders, and front legs), middle (flanks and back), and the rear part of the animal (rump, hind legs, and tail), because the accumulation of skin lesions in different locations has been associated with different types of agonistic behavior [5,22].

#### *2.3. Behavioral Observations*

Infrared cameras, capable of recording in the dark, were installed overhead (Bullet IP cam, Y-cam Solutions Ltd., London, UK). Each camera framed the area occupied by one pen. A total of four cameras were installed, two for each room. Therefore, behavioral data were collected from a subsample of 48 animals, equally balanced between those subjected to darkness, and those kept in a 12 h photoperiod. Unfortunately, it was not possible to identify pigs of different genetic type or gender on videos, so these effects were not considered in the statistical models in which behavioral observations were analyzed.

To avoid interference due to the presence of operators performing the SL count, behavioral data were collected during the 24 h preceding each SL count. The only exception was represented by the behavioral data collected in the first 24 h following the regrouping of pigs, which occurred the day after the first (baseline) SL count. Collection of behavioral data was performed with the Observer software (ver. 12, Noldus Inc., Groeningen, the Netherdlands) on videos recorded on the following days of the trial (Figure 1):

Observations were performed in one interval of 10 min for each of the 24 h of observation days, resulting in a total of 24 intervals per day of observation and 96 intervals per pen for the whole study. During each observation interval, the expression of agonistic behavior by individual pigs was recorded with a focal animal, continuous sampling technique [23]. Agonistic behavior was defined as any interaction involving bites, attempts to bite, physical contact aimed at displacing, or putting off balance another pig (e.g., by pushing one's body against the other, or by putting one's muzzle under the body of the other pig). When such agonistic interaction lasted less than 1 s (i.e., one bite or one attempt to bite not followed by any other agonistic behavior), they were recorded as point events (AGO < 1 s). When the interactions lasted more than 1 s (AGO > 1 s), their duration was recorded, resulting in both number and duration of these interactions being used in statistical analysis.

Since the recording of agonistic behavior was performed for each pig in the pen, an interaction involving only two pigs would result in two recorded episodes of agonistic behavior of identical duration. For interactions involving more than two animals, as many episodes were recorded, as was the number of animals involved; the duration of such episodes, however, could be different since subjects could be involved for a different time in the same interaction (e.g., pig A starts an agonistic interaction with pig B, then pig C is involved along with A and B, then B stops interacting, leaving A and C to continue the interaction; in this case, three episodes of different duration would be recorded).

In addition to agonistic behavior, the number of animals in quadrupedal stance at the beginning of the 10 min observation interval was recorded as a measure of overall activity in the pen within the observation interval (STAND).

#### *2.4. Performance Traits*

In order to evaluate possible long-term effects of the darkness condition provided in the first 48 h following mixing, the main performance traits recorded for pigs in the feeding trial were taken into account. To this purpose, individual BW was collected at the start and at the end of the feeding trial for all the animals (100 pigs and eight pens), whereas individual feed intake (ADFI) was measured daily through the electronic feeder [18]. Data collected were used to compute individual average daily gain during the feeding trial (ADG), average individual daily feed intake, and individual feed efficiency, expressed as gain to feed ratio (G:F).

#### *2.5. Editing and Statistical Analysis*

Total SL count was computed for each pig by summing the SL recorded on the three different locations of the body. Behavioral observations were partitioned in two classes according to the daytime hours in which they occurred (DH: 08:00 to 19:59 h and 20:00 to 07:59 h), which also corresponded to the time at which lights were turned on/off, respectively, with the exception of the first 48 h in the pigs subjected to the dark lighting regimen. After a preliminary analysis aimed at examining their approximation to the normal distribution, data about the SL and the number of agonistic interactions were log transformed prior to statistical analysis.

Log of SL counts were analyzed according to a linear mixed model (SAS 9.4, SAS Institute, Cary, NC, USA) that included the fixed effects of sex (two levels), genetic types (four levels), lighting regime in the first 48 h following mixing (LR, two levels), days of the trial (DAY, four levels), operator (two levels), the DAY × LR interaction, and the random effects of pen within LR and of animal within genetic type and sex. Polynomial contrasts were estimated between the least square means of DAY to examine the response curve of each trait (linear, quadratic, and cubic components) with the advancing of time after mixing. Contrasts between least squares means of LR were estimated separately within each DAY for traits where the LR × DM interaction was significant (*p* < 0.05).

Data concerning the logarithm of the number of AGO < 1 s and AGO > 1 s, the total and mean duration of AGO > 1 s, and the incidence of STAND were analyzed according to a linear mixed model (SAS 9.4, SAS Institute, Cary, NC, USA) that included the fixed effects of LR, days from mixing (DM, four levels), daytime hours (DH, two levels) and the LR × DM and LR × DH interactions, and the random effects of hour of observation within DH and of pen within LR. Polynomial contrasts were estimated between the four least square means of DM to examine the response curve of each trait (linear, quadratic, and cubic components) with the advancing of time after mixing. Contrasts between least squares means of LR were estimated separately within each DH for traits where the LR × DH interaction was significant (*p* < 0.05).

Last, performance traits were analyzed according to a linear mixed model (SAS 9.4, SAS Institute, Cary, NC, USA) that included the fixed effects of sex (two levels), genetic types (four levels), diet (four levels), and LR, and the random effect of pen within LR and diet.

#### **3. Results**

#### *3.1. General Statistics and Skin Lesions Count*

Descriptive statistics of main performance traits recorded on pigs during the whole feeding trial, of SL counts, and of behavioral observation traits during the two weeks following pigs' regrouping are given in Table 1.

At the beginning of the feeding trial, when the pigs were moved to new pens and regrouped, the average BW of pigs approached 87 kg. At the end of the feeding trial, after around 17 weeks on feed, the BW of pigs averaged around 165 kg, which is the target weight for heavy pigs aimed to dry-cured ham production. The ADG was close to 0.66 kg/d and, given an ADFI around 2500 g/d from the start to the end of the trial, the average F:G ratio approached 3.85. The coefficient of variability was below 10% for all performance traits considered except for growth rate, which showed a coefficient of variation close to 14%.

At the fourteenth day following the regrouping procedure, an average total number of 5.24 skin lesions was recorded, with 56% of them being located in the front body area, whereas the middle and rear body area showed a similar SL count distribution. Variation in SL count was high for all the locations in the body.

The average number of AGO > 1 s in the 10 min/h of observation interval approached 2.59, with an average mean duration close to 7 s, whereas on average, 17% of pigs in the pen were standing at the beginning of the 10 min of the observation interval. Additionally, for the behavioral observations, the variation observed was very large.

Average accumulation of SL in the two weeks following pigs regrouping was not influenced by the lighting regime adopted in the first 48 h from mixing (Table 2) independently from the body area. Conversely, the day of trial significantly influenced the distribution of SL irrespective of their position in the body (*p* < 0.0001). As a general trend, the change of log of SL with the advancing of days after regrouping followed a quadratic (SL evaluated in the flanks and back and total sum of SL counts) or cubic (SL evaluated in the front or in the rear part of the body) pattern. Namely, log of SL increased from the day of mixing (Day 0 was considered as a baseline reference for SL) until Day 3 (head, neck, shoulders, and front legs) or 7 from mixing (middle and rear part of the body and total sum of SL counts), whereas it decreased thereafter in all positions of the body.

Moreover, we found a significant (*p* ≤ 0.01) LR × DAY interaction for all the SL counts except for those in the front body area; this suggests that the dynamic of SL accumulation was different according to the lighting regime provided to the pigs during the first 48 h since regrouping. Indeed, pigs kept in the dark room during the first 48 h since regrouping showed a similar pattern of variation since the day of mixing but lower SL accumulation 3 and 7 d following mixing, compared to those pigs provided with a 12 h photoperiod for the whole trial (Figure 2). Differences in SL accumulation between pigs of the two groups were no longer evident 14 d after mixing, with the only exception of skin lesions in the rear part of the body.

**Table 2.** Least squares means of logarithm of skin lesions count (SL) for lighting regimen during the first 48 h after mixing and days of trial.

**Table 2.** *Cont.*

**Figure 2.** Least squares means of logarithm of skin lesions count assessed in: (**a**) front, (**b**) middle, and (**c**) rear position and of (**d**) logarithm of sum of counts at different days from mixing (Days Mix) for pigs kept in dark rooms (solid line) or with a 12 h photoperiod (dotted line) during the first 48 h after mixing (\**p* < 0.05 for difference between means of the two groups within any given day from mixing).

#### *3.2. Behavioral Observations*

Table 3 reports the least squares means of the agonistic behavior variables and of the overall activity, as a function of lighting regimen, days of trial, and daytime hours, as well as the LR × DM and LR × DH interactions on the same variables. No effect was found for the LR or its interaction with DAY on any of the agonistic behavioral variables or the overall activity.

1 Agonistic behavior and activity measured during the first 10 min of each hour.

There were more AGO < 1 during daytime than at night (*p* = 0.001); no change was observed in the number of such interactions as a function of DAY.

There were more agonistic interactions longer than 1 s during daytime than at night, and they were also longer, in terms of average as well as total duration. The mean and total duration of such interactions decreased with increasing days from mixing, and fewer of such interactions occurred as the days from mixing increased.

The incidence of standing pigs on total number of pigs in the pen was affected in a similar manner, with a higher overall activity during daytime than at night, and a decrease of overall activity as a function of days from mixing.

#### *3.3. Performance Traits*

As shown in Figure 3, the lighting regime during the first 48 h following regrouping did not affect growth performance traits of a 17-weeks feeding trial (*p* > 0.10). Pigs of two groups showed similar BW at the end of the feeding trial and comparable growth rate. As average feed intake was nearly identical in pigs provided with 48 h dark or normal photoperiod after mixing, the feed-to-gain ratios of the pigs of the two groups were also very similar.

**Figure 3.** Least squares means of: (**a**) final BW, (**b**) overall average daily gain, (**c**) average daily feed intake, and (**d**) feed-to-gain ratio of pigs given 0 h (grey bar) or 12 h light daily (white bar) during the first 48 h after mixing (*p* value of the differences between means > 0.10).

#### **4. Discussion**

Agonistic behavior expressed when unfamiliar pigs are housed into newly formed social groups has been recognized as a one of the major welfare issues for pig husbandry [1]. Although aggression following mixing of growing or breeding pigs has been widely investigated in the last decades, mitigation strategies proposed seem scarcely implemented in commercial herds, so that the problem has not been reduced yet [3,4]. In the present study, we investigated if the provision of darkness in the 48 h after the formation of new social groups in growing pigs is effective in reducing the expression of agonistic behavior, and its outcome in terms of accumulated skin lesions.

 Moreover, SL are a direct outcome of physical damages due to agonistic interactions, and their frequency and intensity can be related to the level of welfare impairment following regrouping.

In this study, a large variation in the total number of SL was found, similar to Turner et al. [5]. In agreement with the latter and other studies [5,9,24], lesions were mostly concentrated in frontal part of the body. Turner and collaborators [5] suggested that lesions in such region are the outcome of reciprocal active fighting, whereas those accumulated in the medium and rear part of the body are correlated to receiving aggression and retreating.

The accumulation of SL progressively increased over time, from the third to the seventh day of trial for front SL and middle, rear, and total ones, respectively. This pattern is generally consistent with data from the literature, although the schedule of SL evaluation after regrouping is variable. Turner and collaborators [5] observed an increase of SL 24 h post-mixing in the vast majority of pigs evaluated, with a greater increase for front SL compared to the rear body part. Similarly, Wurtz and collaborators [22] reported a steep increase of SL in all body locations 24 h following regrouping, whereas three weeks after regrouping SL decreased and accumulation was generally lower than that scored at regrouping. Stukenborg and collaborators [24] observed an increase in SL evaluated 48 h after regrouping on the front part of the body on the majority of pigs considered; conversely, no positive difference was found for over 60% and 70% of pigs in LS observed in the middle and rear regions, respectively, concluding that their contribution in the prediction of agonistic behavior seemed to be rather insignificant. Differences in the trend of variation of SL found in the present study could corroborate such conclusion, as front SL peaked 3 d after mixing, including the period when agonistic

behavior is particularly intense, whereas SL judged at the medium and rear parts of the body peaked at later times.

Keeping pigs in the dark during the first 48 h since regrouping, compared to those provided with a 12 h photoperiod for the whole trial, did not affect the average accumulation of SL over the two weeks following regrouping or the pattern of variation of SL since the day of mixing; however, it significantly decreased the sum of SL and the accumulation of SL in the medium and rear part of the body in the first 7 d following regrouping, thus mitigating the negative effects of mixing unfamiliar pigs on skin integrity. Barnett and collaborators [16] observed a short-term decrease of aggression in pigs mixed 30 min after sunset compared to pigs mixed in the morning. However, the accumulation of SL three days post-mixing was similar between groups, suggesting that mixing after sunset simply delayed SL rather than really mitigating the effects of regrouping.

With regard to agonistic behavior, its expression was mostly affected by the time of day, with both duration and frequency of agonistic interactions being fourfold higher during daytime than nighttime. Our results are in agreement with previous studies reporting higher peaks in the expression of agonistic interactions in the middle of the day and lower peaks at night following a circadian rhythm [24]. In the present study, this pattern was clearly associated with the pigs' overall activity level, which was also, predictably, lower at night than during daytime. It is worth noting, however, that no difference in the effect of daytime on agonistic behavior or activity levels was found between the two experimental groups. Therefore, the provision of 48 h of darkness did not alter pigs' circadian rhythm, at least in terms of behavior. Although the timing by which photoperiod acts on agonistic behavior has not been investigated before, other physiological traits, such as melatonin concentrations, have been shown to closely follow abrupt changes in photoperiod; nonetheless, even melatonin secretion may take up to one week to fully entrain after a new photoperiod is instated [25]. Thus, our findings are well consistent with a lack of relevant effects in the provision of 48 h of darkness on circadian rhythm.

Agonistic behavior was also affected by the time elapsed from the day of mixing. The effect was clearly evident on the average and total duration of agonistic interaction, which decreased linearly (for average and total duration) or along a quadratic curve (average duration only), from Day 0 to the fourteenth day after mixing. Additionally, the number of agonistic interactions longer than 1 s decreased linearly with the advancing of days of trial, whereas no significant effect of day from mixing was observed on the number of short, instantaneous agonistic events. Therefore, although agonistic behaviors do not cease to occur, they appear to be solved significantly faster over time [26]. Therefore, our results confirm that aggressive behaviors aimed at re-establishing a social hierarchy in the new groups mostly occur during the first 48 h after mixing, as previously reported [6].

Previous studies suggested that impeding access to visual stimuli when groups of unacquainted pigs are newly formed may reduce the expression of agonistic behavior [16]. However, as the aggressions resume at the re-establishment of visibility (end of darkness period), this practice merely delayed the expression of this behavior [16]. In our experiment, the expression of agonistic behavior was not affected by the provision of 48 h of darkness, nor by its interaction with the time from mixing, indicating that agonistic interactions were concentrated during the first days after mixing, and progressively decreased over time, in both groups. This implies that there was no rebound in the frequency or duration of agonistic interactions in animals that had been kept in darkness once visibility was re-established. Our results are in contrast with those reported by Barnett et al. [16], who observed increased agonistic interactions between pigs being mixed before a 12 h (overnight) of darkness as the end of this period coincided with the peak of expression of agonistic behaviors within the group of pigs. On the contrary, in our study, this peak occurred while pigs were still kept in the dark, indicating that the provision of a 48-h period of darkness is sufficient to overcome such an acute agonistic phase; at the same time, the period is long enough to avoid relapses when visual stimuli are again made available. A further implication of the lack of differences between experimental groups in the expression of agonistic behavior over time is that the removal of visual stimuli of conspecifics does not seem to prevent pigs from engaging in agonistic interactions, nor to carry them on for as long as in plain visibility. Thus, at least in the conditions assessed by the present study, other sensory modalities seem sufficient to drive pigs' agonistic behavior. Again, this is in line with previous literature, showing not only a significant role of olfactory [27–30] and pheromonal perception [14,31,32] on agonistic behavior, but also that removal of visual stimuli (e.g., by temporarily blinding the animals, or by covering their heads with hoods) brings no alteration in pigs ability to form and maintain a hierarchy [33,34].