Evaluation of Coat Color Inheritance and Production Performance for Cossbreed from Chinese Indigenous Chenghua Pig Crossbred with Berkshire

Background: Crossbreeding programs have been used extensively to improve the native pig’s overall production performance while maintaining superior meat quality. Chenghua (CH) pig is a traditional Chinese indigenous breed with superior meat quality characteristic but poor growth and carcass traits. In recent years, we implemented Berkshire × Chenghua (BC) crossbreeding scheme and have bred the new crossbreed BC pig through selection for four generations. The present objective was to determine the black coat inheritance and evaluate production performance for crossbreed BC F4 pigs in comparison with those of control purebred CH pigs. Results: Coat color of crossbreed BC pigs exhibits a “dominant black” hereditary pattern. Twelve mutation sites for MC1R gene were identied between “uniform black type” and “domino black spotting type” pigs and all piglets derived from boars or sows genotyped ED1ED1 homozygous for MC1R gene showed uniform black coat phenotype. The crossbreed BC F4 gilts displayed a relatively good reproductive performance, showing a higher litter (total no. born: 12.06 and no. born alive: 11.14) and tear size (13.14), heavier at farrowing litter (11.92 kg) and at weaning litter (65.87 kg) than purebred CH gilts, but they reached puberty later than CH gilts (178.44 vs.125.45). The crossbreed BC F4 pigs exhibited improved growth and carcass characteristics with a higher average daily live weight gain (535.28 g vs. 447.11 g), lower feed-to-gain ratio (3.06 vs. 4.03), and higher carcass lean meat rate (50.76% vs. 42.58%) than purebred CH pigs. Importantly, similar to those of purebred CH pigs, the crossbreed BC F4 pigs produced super meat-quality characteristics, showing ideal pH and meat-color values, high intramuscular fat content (3.24%) and water-holding capacity (drip loss: 1.68%), and acceptable muscle-bre parameters. C18:1, C16:0, C18:0, and C18:2 were the main fatty acids in M. longissimus lumborum in the two breeds, and a very high polyunsaturated/saturated fatty acid ratio of ~0.39 was observed in the BC F4 pigs. Conclusion: These results indicate that crossbreed BC F4 pigs exhibit a uniform black coat pattern and acceptable total production performance, and it can be extensively used in commercial pig production to provide high-quality niche products. meat ratio (~ 50%) and backfat thickness (~ 2.6 cm). The present result is similar to those of previous studies [3, 4, 6], which reported that the carcass characteristics of hybrid pigs were largely improved compared with those of the native pig breeds and a mean of 51 ~ 55% for lean meat ratio was found in the crosses from Duroc × Dahe, Celta × Landrace, Celta × Duroc, and Duroc × Yanan. According to the market demand of black pork in China, we suggest that it is perfectly suitable for black breed to reach a mean of 53 ~ 55% for lean meat ratio (about 3 ~ 5% increase). So, to achieve an ideal lean meat ratio, we will select back fat further down to 20 mm for alive back fat thickness at 180 d age in the subsequent breeding process of BC pigs.


Background
The Chenghua (CH) pig is a traditional black breed native to South-Western China in the Sichuan province and it is characterized by superior meat quality traits and good adaptability for extensive and management [1]. However, owing to the undesirable traits such as slow growth rate and low lean meat percentage [2], the production system of purebred CH pigs has been almost displaced and CH pigs have been included in the National Program for Farm Animal Resources since 2014.
Crossbreeding programs have been used extensively to improve the native pig's overall production performance while maintaining superior meat quality for F1 hybrid pigs from Duroc × Dahe, Duroc or Landrace × Celta, Duroc × Korean Native Black Pig, and Duroc × Berkshire × Yanan [3][4][5][6]. However attribution to the break-up of epistatic complexes since F2 generation [7,8], improving and stabilizing the obtained heterosis basing on breed additive and dominance effects is a considerable challenge for a new breed formation arisen from the cross of two or more existing breeds.
In recent years, so as to utilize the genetic resource of CH pig to improve its overall production performance and produce superior pork, we have implemented the crossbreeding scheme of Berkshire (BS) × CH (BC) and bred the new crossbreed BC pig through selection for four generations.
Nowadays, the core breeding group of BC pigs contains 30 unrelated boar strains and more than 1000 sows, and the production system of BC pigs can supply approximate 50,000 black fattening pigs per year to meet the market demand for high quality pork. However, there is no scienti c data evaluating the production performance as well as coat color variation for the new crossbreed BC pigs. Therefore, the present objective was to determine the black coat inheritance and evaluate production performance for crossbreed BC F4 pigs in comparison with those of control purebred CH pigs.

Materials And Methods
Breeding group structure and management All pigs were kept on the Qionglai Jialin Ecological Farm, Chengdu city, China. The core breeding group of purebred CH pigs included 8 unrelated boar strains and 320 sows. The BC crossbreed base population derived from the progeny of 10 Berkshire (BS) unrelated boar strains and 218 CH sows with above synthetic selection index. Then the more advanced generations were bred by the method of population subgeneration breeding, and the cross breeding population included 30 unrelated boar strains and about 600 sows in each generation. Mating was done in a way that reduced inbreeding. Animals with a relationship coe cient of above 5% were not mated to each other. All matings were through arti cial service. The selection of boars and gilts was mainly based on the paternal and matrilineal selection index, respectively; meanwhile, the selection also was in combination with pedigree and phenotypic characteristics. The management and feeding conditions of all pigs at different stages of production were largely designed to the conditions that are experienced in modern breeding areas. The diets met the National Research Council (NRC) (2012) recommendations for the different production phases [9].
Observation of coat color variation and collection of reproductive performance data Coat color was observed for cross piglets per litter and their parents, and "the uniform black" or "domino black spotting" phenotypes were recorded for more than 16,000 pigs. The reproductive performance for 927 crossbreed BC F4 gilts and 320 purebred CH gilts were recorded and collected from January 2019 to July 2020, respectively. Number of teat was recorded for gilts, meanwhile the puberty of gilts was de ned as the rst observed estrus followed by a second estrus approximately 21 or 42 d later. The total number of pigs born and number of pigs born alive per litter were recorded, meanwhile piglets per litter were weighed within 12 h of birth and at 28 d of age for litter weight at birth and litter weight at weaning, respectively.

Identi cation of MC1R SNPs
Hairs with follicles were collected after disinfection with 75% alcohol at the shoulder and then were washed twice with PBS and stored in a refrigerator at -20℃. Genomic DNA was extracted according to the instructions using a DNA extraction kit (Magen, Guangzhou, China). Two pairs of primers were designed according to MC1R reference sequence (GenBank accession number FJ6655467.1) to amplify the complete MC1R DNA sequence ( Table 1). The PCR reaction system was 25µL, containing 22µL 2 × TsingKE Master Mix (TsingKE, Beijing, China), 1µL upstream primer, 1µL downstream primer, and 1µL DNA. Thermocycling conditions began with a denaturing at 98 °C for 2 min, followed by 34 cycles of denaturing at 98 °C for 10 s, annealing at the Tm (Table 1) for 10 s, and extension at 72 °C for 10 s, and nally extension step at 72 °C for 5 min, Store at 4 °C. The ampli cation process was carried out using the Genemate Series PCR machine (Analytik Jena, Germany). 5 µl PCR products were used for 1.5% agarose gel electrophoresis to determine whether the MC1R gene was ampli ed. BigDye® Terminator V3.1 was used for sequencing puri cation. 3730 sequencer was used for sequencing and 3730XL was used for data collection. The sequencing sequence and peak graph were obtained by Chromas. The obtained sequence is spliced by CExpress to obtain a complete sequence. The sequence was aligned by Blast in NCBI.

Measurement of fattening and slaughtering performance
In total, 80 pigs (20 castrated males and 20 females for BC F4 or CH pigs, respectively) were randomly selected at about 60 days of age (with weight at about 15 ~ 20 kg). These pigs in each breed were born to a total of 10 litters that were produced by ve sires and 10 sows. All pigs were housed in individual pens (2 m 2 ) located in the same room and were fed twice a day with the same diet, and pigs had ad libitum access to diet and water (nipple drinkers). To gain expected market slaughter weight and age, the fattening experiment lasted for 180 days for BC pigs and 240 days for CH pigs after 7 days of the adaptation period, respectively. The experimental diets met the National Research Council (NRC) (2012) recommendations for the two different growth phases [9]. In the fattening period, the data of initial live weight, nal live weight and feed consumption were recorded to determine daily live weight gain and feed-to-gain ratio.
At the end of the fattening period, 20 pigs (10 castrated males and 10 females) per breed were slaughtered to determine carcass-composition characteristics according to the described methods [3,6]. The measured carcass traits included carcass length, dressing percentage, back fat thickness, loin muscle area, skin thickness, number of ribs, and dissection ratio of bone, muscle, subcutaneous fat and skin. The M. longissimus lumborum of the left side of the carcass at the last third to fourth rib was sampled and used to measure meat quality according to the described methods [3,6]. The measured meat quality traits included pH values, color parameters, water-holding capacity, and muscle-bre parameters. The muscular fatty acid (FA) composition was analyzed using a gas chromatography (Agilent 6820, Agilent Technologies, Palo Alto, CA, USA) and capillary column (HP-Innowax, Agilent, 30 m long, 0.32 mm internal diameter, 0.25 mm lm thickness) according to the described method [10].

Statistical analyses
Statistical testing was implemented by IBM SPSS Statistics 22. The data are quanti ed as the mean ± SEM for one group. The differences of groups were calculated using independent T test. P < 0.05 is considered to be signi cant, *P < 0.05, **P < 0.01, and ***P < 0.001.

Results
Speci c black coat were selected for the crossbreed BC pigs basing on E D1 E D1 homozygous genotype of MC1R gene To discover the hereditary pattern of black coat for the crossbreed BC pigs, we rstly observed the phenotypic changes of coat color in cross generation. As a result, all F1 crosses (3,140), which derived from 10 BS boars (domino black spotting type) and 320 CH sows (black type), were uniform black; however F2 cross pigs (5,906), which derived from 30 F1 black boars and 588 F1 black sows, were black or domino in the proportion of about 3:1. Interestingly, when 26 F2 black boars were used in the production system, F3 cross pigs (2, 038 out of 2,041) derived from 9 F2 black boars (called homozygote) and 182 F2 black sows were uniform black, but F3 cross pigs (3,698) derived from other 17 F2 black boars (called heterozygote) were black or domino in the proportion of about 5:1 ( Table 2). The results indicated that the black coat of crossbreed BC pigs might be controlled by a dominant single gene and it could inherit in accordance with Mendel's law of segregation. , and F3 means the BC cross pigs from rst, second, and third generation, respectively. "-"means that the ratio cannot be calculated or very large because coat color pattern of (nearly) all pigs is black.
Basing on the important regulatory role of melanocortin 1 receptor gene (MC1R) on body melanin deposition [11], we considered MC1R gene as a potential candidate gene for the black coat of crossbreed BC pigs, and cloned and sequenced the complete DNA of MC1R gene for these samples from BS, CH, F1 crosses, F2 black boars (called homozygote or heterozygote), and F2 domino black spotting cross pigs, respectively. As a result, we obtained a 1552 bp DNA sequence of MC1R (GenBank accession number AY960624) and screened out 12 mutation sites in complete DNA sequence of MC1R gene from these samples (Table 3). According to the de nitely established alleles at the MC1R locus [11], we found that the CH pigs and F2 black boars (called homozygote) showed the typical E D1 E D1 homozygous genotype while BS and F2 crosses with domino black spotting showed other opposite E P E P homozygous genotype; meanwhile F1 crosses and F2 black boars (called heterozygote) displayed the same E D1 E P heterozygous genotype ( Table 3). The result indicates that the E D1 allele is associated with a black coat phenotype and inherits in a dominant pattern in the crossbreed BC pigs. According to the above results, we selected these black boars and sows genotyped with E D1 E D1 since F3 generation to reproduce offspring. As expected, all BC F4 cross pigs showed uniform black in whole production system.  Reproductive performance of crossbreed BC gilts compared with purebred CH gilts Table 4 summarized the reproductive performance of crossbreed BC F4 gilts compared with purebred CH gilts. The mean number of teat was higher for BC gilts than for CH gilts (P < 0.001, 13.14 vs. 12.14). The mean age at puberty of BC gilts was 178.44 d although it was older than CH gilts (P < 0.001, 125.45 d). Total number of pigs born (12.06 pigs) and number of pigs born alive (11.14 pigs) per litter were higher (P < 0.001) for BC sows than for CH sows (10.31 pigs and 9.82 pigs, respectively). Breed effects were signi cant for litter birth weight and litter weaning weight. At birth and weaning at 28 d age, litters from BC sows (11.92 kg and 65.87 kg, respectively) were heavier (P < 0.001) than those from CH sows (8.33 kg and 49.4 kg, respectively). Growth and carcass traits of crossbreed BC pigs compared with purebred CH pigs As expected, throughout the fattening period, the crossbreed BC F4 pigs grew faster than that of purebred CH pigs (P < 0.001), with a higher average daily live weight gain (535.28 g vs. 447.11 g ); meanwhile the feed consumption was more e cient for BC crosses than for CH pigs (P < 0.001, feed-to-gain ratio: 3.06 vs. 4.03) ( Table 5).

Discussion
Black coat variation of pig being associated with MC1R gene Coat color is an important characteristic of various pig breeds and color variations may be useful in identifying the components of some speci c crossbreeding schemes as well as contributing to the image associated with high quality regional products [12]. Here, the observation dealing with crossbreeding experiments between the Chinese indigenous CH pigs (uniform black) and imported BS pigs (domino black spotting) exhibits a "dominant black" coat color hereditary pattern. Similar result was reported that the allelism between "uniform black type" and "domino black spotting type" may also be inferred from Large Black × Berkshire cross pigs [13].
Our observed segregation results prompted the important discovery that the coat color variation of crossbreed BC pigs is determined by the single MC1R gene although more than eight color loci have been discovered [14]. Twelve mutations were screened for MC1R gene in BC crosses, which represent two typical E D1 and E P alleles inferred according to the previous report [9]. Our result indicates that the E D1 allele associated with a black coat phenotype inherits in a dominant pattern in the crossbreed BC pigs. Consequently, we have selected these the black boars and gilts genotyped with homozygous E D1 E D1 since F3 progeny, and largely succeeded in the BC breed standard of black coat.
Crossbreeding improving sow reproductive performance Level of sow productivity is one of the most importance production traits affecting the e ciency of a swine enterprise [15]. Crossbreeding programs have been used extensively to improve reproduction by exploiting breed additive effects, breed maternal effects, and heterosis. Young (1995) reported that Chinese indigenous breed Meishan, Fengjing, and Minzhu pigs can be used to produce crossbreed gilts that have a higher level of reproductive performance than Duroc crossbreed gilts [16]. Here, we found that the crossbreed BC F4 gilts have a higher litter and tear size, and meanwhile the BC gilts also are heavier at farrowing and at weaning than purebred CH gilts. Noteworthy, a mean of 12.1 pigs for total litter size and 11.4 pigs for alive litter size of BC sows has an advantage in litter size during the breeding process. Similar result also was reported that the cross sows from the Chinese native Meishan, Fengjing and Minzhu pigs show a total number of pigs born (12.0 to 11.0 pigs) and number of pigs born alive (11.3 to 10.7 pigs) per litter [14].
A favorable mean age at puberty of 178 d for crossbreed BC gilts was found although BC gilts reached puberty later than CH gilts. Similar result was reported that a mean age at puberty of 118 and 217 d for purebred Meishan and its crossbred gilts [17]. However, purebred Duroc averaged 234 d at puberty, compared with 210, 205, and 201 d for Hampshire, Pietrain, and Spot pigs [18].
Crossbreeding improving growth performance and carcass composition Previous reports revealed that the growth performance of hybrid pigs from Duroc × Dahe, Celta × Landrace, Celta × Duroc, and Duroc × Yanan was largely improved compared with that of native pig breeds [3,4,6]. Here, two important growth traits including weight gain and feed e ciency are largely improved in crossbreed BC F4 pigs, indicating that the BC pigs reach a competitive slaughter age (about 200 d) at above 100 kg slaughter weight.
Meanwhile, the crossbreed BC pigs exhibit an improved carcass characteristic such as moderate lean meat ratio (~ 50%) and backfat thickness (~ 2.6 cm). The present result is similar to those of previous studies [3,4,6], which reported that the carcass characteristics of hybrid pigs were largely improved compared with those of the native pig breeds and a mean of 51 ~ 55% for lean meat ratio was found in the crosses from Duroc × Dahe, Celta × Landrace, Celta × Duroc, and Duroc × Yanan. According to the market demand of black pork in China, we suggest that it is perfectly suitable for black breed to reach a mean of 53 ~ 55% for lean meat ratio (about 3 ~ 5% increase). So, to achieve an ideal lean meat ratio, we will select back fat further down to 20 mm for alive back fat thickness at 180 d age in the subsequent breeding process of BC pigs.

Breed affecting meat quality characteristics
Meat quality is an important factor affecting how pork can be utilized. When choosing the best crossbreeding strategy, it is important to recognize pig breeds determine meat quality traits [19]. In this study, the crossbreed BC and purebred CH pigs produce excellent meat-quality characteristics, which showing normal and high pH values referred to the recommended normal levels (pH 1 > 6.1 and pH 2 : 5.5 ~ 6.0) [20], normal and low meat color parameters according to NPPC (2000) standards (Minolta L-value levels of 37-49) [21], lower drip loss than those for foreign breeds with above 3% [22], and smaller muscle-bre areas than did foreign hybrid pigs with above 5,000 µm 2 [19].
As the most important parameter of meat quality, The IMF content is related to the organoleptic characteristics of pig meat and in uences meat and meat-product quality [23]. An IMF content of 2 ~ 3% is suggested to be optimal for food quality [24,25]. Interestingly, the crossbreed BC pigs in the present study exhibited relatively high IMF content (3.24%). Meanwhile, a higher PUFA:SFA ratio of IMF leads to better digestion rates and an improved digestibility of SFAs with emulsifying agents [26,27] and the recommended PUFA:SFA ratio is more than 0.4 [28]. Here, a similar PUFA:SFA ratio of ~ 0.39 also was found in the BC pigs. The BC pig meat with high IMF content and PUFA:SFA ratio can meet the demand for high-quality niche pork products.
The super meat quality properties for the crossbreed BC pigs might be attributed to the breed attributes of their parent. Because previous studies found that the Berkshire sire pigs are superior for loin meat and eating [22], and these characteristics are, consequently, thought to attribute to its higher overall likeability score [29,30] and improved acceptability compared with European commercial pork breeds [31]. Meanwhile, the present result and previous study indicate that the CH pigs also is characterized by superior meat quality traits [1].

Conclusions
The coat color of Berkshire × Chenghua (BC) cross pigs exhibits a "dominant black" phenotypic hereditary pattern and the new crossbreed BC F4 pigs exhibit a uniform black coat pattern by selecting their sire and maternal pigs with E D1 E D1 h omozygous genotype for MC1R gene. Meanwhile, the crossbreed BC F4 pigs have an outstanding overall production performance, which showing that BC pigs have a relatively good maternal reproductive performance, market-competitive improved growth and carcass characteristics, and super meat-quality characteristics. These results indicate that the new crossbreed BC black pigs can be extensively used in commercial pig production to provide high-quality niche products.