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Abstract [Objectives] This study was conducted to illustrate the possible effects of heredity on the growth， reproduction and immune traits of Enshi Black pig.
[Methods] The main growth， reproduction and immune traits of three populations （Goutou type， Shizitou type and an intermediate type between them） of Enshi Black pig were surveyed， and the correlations among these traits were analyzed.
[Results] The health score of pregnant sows， average litter size and weight gain of piglets of intermediate type were all very significantly higher than those of Goutou and Shizitou types （P<0.01）. The percentage of lymphocytes of intermediate type was significantly higher than that of Goutou and Shizitou types （P<0.05）. The number of white blood cells （WBCs） and the number of neutrophils of intermediate type very were significantly higher than those of the other two types （P<0.01）. The percentages of CD3+CD4+ double positive T cells （helper/inducer T lymphocytes） and CD3+CD8+ double positive T cells （suppressor/cytotoxic T lymphocytes） and the CD4+/CD8+ ratio （the ratio of helper T cells/suppressor T cells） of intermediate type were all significantly higher than those of the other two types. Correlation analysis showed that there was a very significant positive correlation between health score and litter size （P<0.01， R=0.336）， a significant negative correlation between the percentage of lymphocytes and the number of neutrophils （P<0.05， R=-0.733）， and a significant correlation between the percentage of CD3+CD8+ T cells and the number of platelets （P<0.05， R=0.700）.
[Conclusions] The intermediate type Enshi Black pig inherits the excellent traits of both Goutou type and Shizitou types， and thus has better growth， reproduction and immune traits than the other two types.
Key words Enshi Black pig; Intermediate type; Health score; Growth traits; Reproductive traits; Immune traits
As a major breed of black pigs in western Hubei， China， Enshi Black pig adapts well to mountainous and hilly areas. Its meat is of high quality， bright red， tender and juicy， can be used for the production of highquality hams and smoked bacons[1-5]. There are three major populations of Enshi Black pig： Shizitou （large size）， Ermei （medium size） and Goutou type （small size）[2].
By investigating the fattening performance of Enshi Black pigs under different nutrient levels， Deng et al.[4] found that the body weight of Enshi Black pig increased at 509 g per day and reached 92 kg at eight months of age， when they were raised according to standard growingfattening feeding programs in South China; the average daily weight gain was 392 g when their feed was reduced to only 70% of the standard， and the body weight of 8monthold piglets was 75 kg. Three hybrid populations （intermediate type， Hei Bai and Hong Bai） of Shizitou and Goutou type Enshi Black pigs were generated through sib mating and backcrossing by the Black Pig Research Institute of Yangtze University in the past five years. Among them， the intermediate type Enshi Black pig has stable traits： a proportionally shorter rostrum， straight face， less back hair， medium size and gentle temperament， and is considered as a new breed. However， the disease resistance of different populations of Enshi Black pig， especially the correlations between disease resistance and other traits of these populations are still unclear. Therefore， this study was conducted to investigate and compare the health index， reproductive traits， growth traits and immune indices of Goutou， Shizitou and intermediate populations of Enshi Black pig， to illustrate the possible effects of heredity on the main growth traits and disease resistance of black pig. The results may provide a theoretical basis for the detection and identification of genetic variation and conservation of local pig breeds in China.
Materials and Methods
Materials
Animals
Fiftynine healthy Enshi Black sows （weighing about 70 kg each， including 14 of Goutou type， 16 of Shizitou type and 29 of intermediate type） and one Duroc boar were selected from the Experimental Base of the College of Animal Science， Yangtze University and used in this experiment.
Feed
The main ingredients of the pigs feed include maize flour， fish meal， wheat flour， wheat middlings， soybean meal， wheat bran， limestone powder， calcium hydrogen phosphate， copper sulfate， ferrous sulfate， zinc sulfate， manganese sulfate， vitamins （VA， VD3， VE， niacin， calcium pantothenate， etc.）， lysine， methionine， threonine， phytase and alanine.
In addition， 4 mg/kg flavomycin and 100 mg/kg olaquindox were added to the pigs diet， with a withdrawal period of 35 days.
Reagents
Phosphatebuffered saline （PBS）， peripheral blood lymphocyte （PBL）， red blood cell （RBC） Lysis buffer， fluoresceinlabeled monoclonal antibodies （McAbs） against CD3/CD4/CD8 were all purchased from Becton， Dickinson and Company， USA.
Instruments
Digital scale （Kunshan Tuoheng Electromechanical Co.， Ltd.）， DSCW800/BC CN2 digital camera （Sony）， BC46S incubator （Hisense， Beijing）， sampling box （Taizhou Boyurun Experimental Equipment Co.， Ltd.）， EDTAK2 vacuum blood collection tube （Jiangsu Kangjian Medical Apparatus Co.， Ltd.）， glass slides， FACSort flow cytometer （BD， USA） and XE2100 automated hematology analyser （Sysmex） were the main instruments used in this study. Feeding administration
All pigs were raised in separate pens， with one pig in each pen throughout the entire experimental period. They were given sufficient amount of feed and had free access to sterile water. All the fresh vegetables and fruits were washed， and disinfected before they were provided to the animals. Each piglet was weighed and ear notched within the first 24 hours of birth. The piglets were taildocked and teethclipped within the first four days of birth， and weaned at 28 days of age. The pigs used in the test were raised by the same breeders in two connected pig houses， and their living conditions， nutritional levels and immunization procedures were all the same.
Methods
Scoring of health status of pregnant sows
The 59 healthy Enshi sows of the three groups were all mated to the same Duroc boar during three successive reproductive cycles. Each sow was scored on a 10score scale according to the health status of the sows and piglets from the three litters： 0.5 points were deducted for each occurrence of clinical mastitis， 0.5 points for each birth problem （such as mucopurulent discharge， metritis， pyometra， abnormal vaginal discharge， ovarian cysts， placental retention and miscarriage）， 1 point for deafness， 0.5 points for diarrhea in suckling piglets， and 0.5 points for poor growth of piglets. Another 2 points were deducted if medical therapy did not work.
Average litter size of three births
The sows were mated with the same Duroc boar for three successive births. The gestation period， litter size， number of piglets born alive per litter， number of surviving piglets per litter of each Enshi Black sow were measured， and the health status of each piglet was surveyed. The piglets were defined as nondefective or defective according to their weight， growth potential， health status， etc. Average litter size was defined as the average number of weaned piglets of the three births per sow.
Birth weight and weaning weight of piglets
The piglets were weighed on the day of birth， at 28 days of age when they were weaned and at late weaning stage. Then the birth weight per litter， weaning weight per litter and weight gain per litter were calculated.
Blood test
Blood sample was collected from the marginal ear vein of each sow， and analyzed with 2 h using XE2100 automatic hematology analyzer （Sysmex）， following manufactures instruction.
Flow cytometry
Blood sample was collected into BD VacutainerTM plastic blood collection tubes with sodium heparin from the marginal ear vein of each sow after farrowing. The lymphocyte subsets in the blood samples were then separated by flow cytometry. The samples were pretreated following the flow cytometry assay kit instructions. In detail， 2 ml of each blood sample was mixed with 2 ml of Hanks Balanced Salt Solution. Then， 4 ml of lymphocyte separation medium was slowly added along the tube wall to above diluted blood sample and allowed to stand for 10 min， centrifuged at 1 500 r/min for 15 min. Then 1.5 ml of the liquid in the middle peripheral blood mononuclear cell （PBMC） layer was collected and centrifuged at 3 000 r/m for 30 min. The supernatant was discarded， 500 μl of red blood cell lysate was added to the cell pellet， allowed to stand for 10 min， mixed by shaking. After that， the sample was centrifuged at 1 000 r/min for 2 min. The PBMCs pellet was resuspended in 600 μl of PBS buffer， added with 5 μl of CD3+ antibody， 5 μl of CD4+ antibody and 2 μl of CD8+ antibody and incubated on ice bath at 4 ℃ for 15 min before the sample was assayed by flow cytometry. Statistical analysis
Analysis of variance， significance test， and correlation analysis were done using SPSS19.0. T test was carried out to determine the difference in immune traits between different Enshi Black pig populations. The correlations between different traits was calculated through bivariate analysis. Bonferroni correction was used to counteract the problem of multiple comparisons.
Results and Analysis
Comparison of reproductive traits among Enshi Black pig populations
Health score
As shown in Fig. 1， the health score of intermediate type pigs stabilized at above 8 points， while that of Shizitou and Goutou types fluctuated greatly， and the lowest score was 5 points. There was very significant difference in health score between intermediate type and the other two types （P=0.001 4<0.01）.
Average litter size
As shown in Fig. 2， the average litter size of the three births for the intermediate type pigs fluctuated slightly around 10， while that of Shizitou and Goutou pigs fluctuated greatly， and the smallest average litter size was 4. There was extremely significant difference in average litter size between intermediate type and the other two types （P=0.002 1<0.01）， indicating that the intermediate type pigs are more stable in reproductive performance than the other two types of Enshi Black pig.
Average daily weight gain per litter
As shown in Fig. 3， the average daily weight gain per litter of intermediate type was very significantly higher than that of the other two types （P=0.002 2<0.01）.
Comparison of immune traits among Enshi Black pig populations
Analysis of asymmetric data revealed that the total number of white blood cells （WBCs） of intermediate type showed no significant difference from that of Shizitou type （Table 1）， but was very significantly higher than that of Goutou type. The percentage of lymphocytes of intermediate type was significantly higher than that of Shizitou type， and very significantly higher than that of Goutou type. The number of neutrophils of intermediate type was significantly higher than that of the other two types. There was no significant difference in the number of red blood cells and the number of platelets between the three populations of Enshi Black pig.
As can be seen from Table 2， the percentage of CD3+CD4+ T cells， the percentage of CD3+CD8+ T cells and CD4+/CD8+ ratio of the intermediate type were all significantly higher than those of the other two types， indicating that the intermediate type Enshi Black pig has higher immunological activity than the other two types. Correlation analysis on the traits of the three Enshi Black pig populations showed that there was an extremely significant positive correlation between health score and litter size （P<0.01）， an extremely significant negative correlation between litter size and CD4+/CD8+ ratio （P<0.01）. There was a significant negative correlation between litter weight and the number of red blood cells， the percentage of CD3+CD4+ T cells and the number of neutrophils， the percentage of CD3+CD8+ T cells and CD4+/CD8+ ratio， the number of white blood cells and the percentage of lymphocytes. The percentage of lymphocytes and the number of neutrophils （P<0.05）; the percentage of CD3+CD8+ T cells was significantly positively correlated with the number of platelets， the number of white blood cells and the number of neutrophils （P<0.05）. The results further proved that the health， reproduction and immune traits of the intermediate type Enshi Black pig are all better than those of the other two types.
Conclusions and Discussion
In our experiment， the health status of three Enshi Black pig populations during pregnancy was scored by referring to previous study[6]， and the results showed that the health score of the intermediate type pigs stabilized at above 8， while that of the other two types fluctuated noticeably， and the lowest score was 5， indicating that there was an extremely significant difference in the health status between intermediate type and the other two types of Enshi Black pig （P=0.001 4<0.01）. It can be concluded that the health status of the intermediate type pigs is more stable than the other two Enshi Black pig populations during pregnancy， which proves that the intermediate type has higher disease resistance than the other two types.
Deng et al.[4] evaluated the reproductive performance of different Enshi Black pig populations， and found that the litter size of largesized sows （12.26±2.68 piglets per litter） was significantly higher than that of smallsized sows （only 8.62±1.50 piglets per litter）. The study of Huang et al.[7] showed that average litter was 9.96 for Yorkshire sows， 9.52 for Changbai sows and 9.13 for Duroc sow， showing no significant difference among them （P>0.05）. Fu et al.[8] reported that the average litter size of Changbai， Yorkshire and Duroc sows was 10.07， 9.34 and 10.06， respectively， and the number of piglets born alive per litter was 9.26， 8.87 and 9.85. Our results showed that the average litter size of the intermediate type pigs fluctuated slightly at about 10， while that of the other two types fluctuated greatly， with the smallest litter size at only four. There was an extremely significant difference in average litter size among the three Enshi Black pig populations （P=0.002 1<0.01）. These results revealed that the intermediate type of Enshi Black pig has more stable reproductive performance than the other two types. Peng et al.[5] reported that Enshi Black pig grows slowly and reaches a body weight of about 95 kg at nine months of age. The study of Zhang[9] showed that Large White pig has an average birth weight of 1.5 kg， and reaches a body weight of 6.86 kg at 21 days of age. There are also some studies showing that the weaning weight of piglets can be increased to some extent by changing the ambient temperature， the diet of pregnant sows and suckling piglets[10-11]. Our results showed that the average weaning weight of the intermediate type Enshi Black piglets at 28 days of age was 6.27 kg， which was very significantly higher than that of Goutou and Shizitou Enshi Black piglets （P=0.002 2<0.01）， indicating that the intermediate type Enshi Black pig has stable reproductive and growth performance， which may also prove that the intermediate type sows produce much more milk than the other two types under the same feeding condition. In conclusion， the intermediate type Enshi Black pig has better growth traits than the other two types under the same feeding condition.
White blood cells （WBCs） including lymphocytes and phagocytes， are the cells of the immune system that fight disease by making antibodies or engulfing germs[12， 13]. Lymphocytes are directly involved in both antibodymediated and cellmediated immune responses[14]. As nonspecific immune cells， neutrophils can engulf senescent cells and pathogenic microorganisms， and account for a large proportion （65%-75%） of blood WBCs[15]. When inflammation occurs， neutrophils are attracted to the site of inflammation by chemotactic substances released from damaged tissues， release large amounts of lysosomal enzymes， engulf and digest the microorganisms and tissue debris， preventing the spread of pathogenic microorganisms in the body[16-22]. Our results showed that intermediate type had an extremely significant difference from Goutou type， but no significant difference from Shizitou in the number of WBCs. The percentage of lymphocytes of intermediate type was very significantly higher than that of the other two types. The number of neutrophils of lymphocytes of intermediate type was very significantly higher than that of Goutou type， but showed no significant statistical difference from that of Shizitou， suggesting that the main immune traits of the intermediate type Enshi Black pig may be primarily inherited from Shizitou type， and is better than those of Goutou type. To sum up， the immune traits of intermediate type Enshi Black pig are better than those of Goutou and Shizitou types， which can be explained by heterosis between the two breeds. The size and ratio of T cell subpopulations are important indices of the bodys immune function， and the changes in the population and function of T cell subsets will lead to the disorders in the immune system， and subsequent pathological changes[23]. There are two major classes of surface markers of T lymphocytes： one is CD3 marker， which is shared by all T lymphocytes， the other class is subpopulationspecific： CD4 is expressed in helper/inducer T cells， and CD8 is present on suppressor/cytotoxic Tcells. Elevated CD4+/CD8+ Tcell ratio indicates increased ratio of helper T cells and enhanced immune response[24-27]. By analyzing the CD3+CD4+ and CD4+CD25+ lymphocyte subsets of pigs with low immune response to fever vaccine， Wang et al.[28] found that decreased population of CD3+CD4+ lymphocyte subset and increased population of CD4+CD25+ lymphocyte subset in peripheral blood may result in a low immune response to fever vaccine. After conducting a series of studies on the effects of porcine epidemic diarrhea virus on the immune function of dendritic cells， Gao[29] found that the population of CD3+， CD4+ and CD8+ T cells in the lamina propria and villi of the small intestine significantly increased in the piglets immunized with inactivated porcine epidemic diarrhea virus （PEDV） vaccine and with inactivated K88 48 h later. In the present study， we analyzed the lymphocyte subsets of three Enshi Black pig populations， and found that the percentage of CD3+CD4+ T cells， the percentage of CD3+CD8+ T cells， and CD4+/CD8+ ratio of the intermediate type Enshi Black pig were all significantly higher than those of the other two types， which further indicates that the specific immune system of the intermediate type Enshi Black pig is increased compared to the other two types.
Spearman rank correlation analysis showed that there is a significant positive correlation between health score and litter size， the percentage of CD3+CD8+ T cells and the number of platelets. In addition， we also found that there is an extremely significant negative correlation between litter size and CD4+/CD8+ ratio. There is a negative correlation between the number of red blood cells and the weight gain of weaned piglets. There are many other factors influencing the immunity of pregnant sows， which need to be studied in future work. In summary， the intermediate type inherits the excellent traits of both Goutou and Shizitou types， and thus has better growth， reproduction and immune traits. The results may provide a theoretical basis for the detection and identification of genetic variation and conservation of local pig breeds in China. References
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[Methods] The main growth， reproduction and immune traits of three populations （Goutou type， Shizitou type and an intermediate type between them） of Enshi Black pig were surveyed， and the correlations among these traits were analyzed.
[Results] The health score of pregnant sows， average litter size and weight gain of piglets of intermediate type were all very significantly higher than those of Goutou and Shizitou types （P<0.01）. The percentage of lymphocytes of intermediate type was significantly higher than that of Goutou and Shizitou types （P<0.05）. The number of white blood cells （WBCs） and the number of neutrophils of intermediate type very were significantly higher than those of the other two types （P<0.01）. The percentages of CD3+CD4+ double positive T cells （helper/inducer T lymphocytes） and CD3+CD8+ double positive T cells （suppressor/cytotoxic T lymphocytes） and the CD4+/CD8+ ratio （the ratio of helper T cells/suppressor T cells） of intermediate type were all significantly higher than those of the other two types. Correlation analysis showed that there was a very significant positive correlation between health score and litter size （P<0.01， R=0.336）， a significant negative correlation between the percentage of lymphocytes and the number of neutrophils （P<0.05， R=-0.733）， and a significant correlation between the percentage of CD3+CD8+ T cells and the number of platelets （P<0.05， R=0.700）.
[Conclusions] The intermediate type Enshi Black pig inherits the excellent traits of both Goutou type and Shizitou types， and thus has better growth， reproduction and immune traits than the other two types.
Key words Enshi Black pig; Intermediate type; Health score; Growth traits; Reproductive traits; Immune traits
As a major breed of black pigs in western Hubei， China， Enshi Black pig adapts well to mountainous and hilly areas. Its meat is of high quality， bright red， tender and juicy， can be used for the production of highquality hams and smoked bacons[1-5]. There are three major populations of Enshi Black pig： Shizitou （large size）， Ermei （medium size） and Goutou type （small size）[2].
By investigating the fattening performance of Enshi Black pigs under different nutrient levels， Deng et al.[4] found that the body weight of Enshi Black pig increased at 509 g per day and reached 92 kg at eight months of age， when they were raised according to standard growingfattening feeding programs in South China; the average daily weight gain was 392 g when their feed was reduced to only 70% of the standard， and the body weight of 8monthold piglets was 75 kg. Three hybrid populations （intermediate type， Hei Bai and Hong Bai） of Shizitou and Goutou type Enshi Black pigs were generated through sib mating and backcrossing by the Black Pig Research Institute of Yangtze University in the past five years. Among them， the intermediate type Enshi Black pig has stable traits： a proportionally shorter rostrum， straight face， less back hair， medium size and gentle temperament， and is considered as a new breed. However， the disease resistance of different populations of Enshi Black pig， especially the correlations between disease resistance and other traits of these populations are still unclear. Therefore， this study was conducted to investigate and compare the health index， reproductive traits， growth traits and immune indices of Goutou， Shizitou and intermediate populations of Enshi Black pig， to illustrate the possible effects of heredity on the main growth traits and disease resistance of black pig. The results may provide a theoretical basis for the detection and identification of genetic variation and conservation of local pig breeds in China.
Materials and Methods
Materials
Animals
Fiftynine healthy Enshi Black sows （weighing about 70 kg each， including 14 of Goutou type， 16 of Shizitou type and 29 of intermediate type） and one Duroc boar were selected from the Experimental Base of the College of Animal Science， Yangtze University and used in this experiment.
Feed
The main ingredients of the pigs feed include maize flour， fish meal， wheat flour， wheat middlings， soybean meal， wheat bran， limestone powder， calcium hydrogen phosphate， copper sulfate， ferrous sulfate， zinc sulfate， manganese sulfate， vitamins （VA， VD3， VE， niacin， calcium pantothenate， etc.）， lysine， methionine， threonine， phytase and alanine.
In addition， 4 mg/kg flavomycin and 100 mg/kg olaquindox were added to the pigs diet， with a withdrawal period of 35 days.
Reagents
Phosphatebuffered saline （PBS）， peripheral blood lymphocyte （PBL）， red blood cell （RBC） Lysis buffer， fluoresceinlabeled monoclonal antibodies （McAbs） against CD3/CD4/CD8 were all purchased from Becton， Dickinson and Company， USA.
Instruments
Digital scale （Kunshan Tuoheng Electromechanical Co.， Ltd.）， DSCW800/BC CN2 digital camera （Sony）， BC46S incubator （Hisense， Beijing）， sampling box （Taizhou Boyurun Experimental Equipment Co.， Ltd.）， EDTAK2 vacuum blood collection tube （Jiangsu Kangjian Medical Apparatus Co.， Ltd.）， glass slides， FACSort flow cytometer （BD， USA） and XE2100 automated hematology analyser （Sysmex） were the main instruments used in this study. Feeding administration
All pigs were raised in separate pens， with one pig in each pen throughout the entire experimental period. They were given sufficient amount of feed and had free access to sterile water. All the fresh vegetables and fruits were washed， and disinfected before they were provided to the animals. Each piglet was weighed and ear notched within the first 24 hours of birth. The piglets were taildocked and teethclipped within the first four days of birth， and weaned at 28 days of age. The pigs used in the test were raised by the same breeders in two connected pig houses， and their living conditions， nutritional levels and immunization procedures were all the same.
Methods
Scoring of health status of pregnant sows
The 59 healthy Enshi sows of the three groups were all mated to the same Duroc boar during three successive reproductive cycles. Each sow was scored on a 10score scale according to the health status of the sows and piglets from the three litters： 0.5 points were deducted for each occurrence of clinical mastitis， 0.5 points for each birth problem （such as mucopurulent discharge， metritis， pyometra， abnormal vaginal discharge， ovarian cysts， placental retention and miscarriage）， 1 point for deafness， 0.5 points for diarrhea in suckling piglets， and 0.5 points for poor growth of piglets. Another 2 points were deducted if medical therapy did not work.
Average litter size of three births
The sows were mated with the same Duroc boar for three successive births. The gestation period， litter size， number of piglets born alive per litter， number of surviving piglets per litter of each Enshi Black sow were measured， and the health status of each piglet was surveyed. The piglets were defined as nondefective or defective according to their weight， growth potential， health status， etc. Average litter size was defined as the average number of weaned piglets of the three births per sow.
Birth weight and weaning weight of piglets
The piglets were weighed on the day of birth， at 28 days of age when they were weaned and at late weaning stage. Then the birth weight per litter， weaning weight per litter and weight gain per litter were calculated.
Blood test
Blood sample was collected from the marginal ear vein of each sow， and analyzed with 2 h using XE2100 automatic hematology analyzer （Sysmex）， following manufactures instruction.
Flow cytometry
Blood sample was collected into BD VacutainerTM plastic blood collection tubes with sodium heparin from the marginal ear vein of each sow after farrowing. The lymphocyte subsets in the blood samples were then separated by flow cytometry. The samples were pretreated following the flow cytometry assay kit instructions. In detail， 2 ml of each blood sample was mixed with 2 ml of Hanks Balanced Salt Solution. Then， 4 ml of lymphocyte separation medium was slowly added along the tube wall to above diluted blood sample and allowed to stand for 10 min， centrifuged at 1 500 r/min for 15 min. Then 1.5 ml of the liquid in the middle peripheral blood mononuclear cell （PBMC） layer was collected and centrifuged at 3 000 r/m for 30 min. The supernatant was discarded， 500 μl of red blood cell lysate was added to the cell pellet， allowed to stand for 10 min， mixed by shaking. After that， the sample was centrifuged at 1 000 r/min for 2 min. The PBMCs pellet was resuspended in 600 μl of PBS buffer， added with 5 μl of CD3+ antibody， 5 μl of CD4+ antibody and 2 μl of CD8+ antibody and incubated on ice bath at 4 ℃ for 15 min before the sample was assayed by flow cytometry. Statistical analysis
Analysis of variance， significance test， and correlation analysis were done using SPSS19.0. T test was carried out to determine the difference in immune traits between different Enshi Black pig populations. The correlations between different traits was calculated through bivariate analysis. Bonferroni correction was used to counteract the problem of multiple comparisons.
Results and Analysis
Comparison of reproductive traits among Enshi Black pig populations
Health score
As shown in Fig. 1， the health score of intermediate type pigs stabilized at above 8 points， while that of Shizitou and Goutou types fluctuated greatly， and the lowest score was 5 points. There was very significant difference in health score between intermediate type and the other two types （P=0.001 4<0.01）.
Average litter size
As shown in Fig. 2， the average litter size of the three births for the intermediate type pigs fluctuated slightly around 10， while that of Shizitou and Goutou pigs fluctuated greatly， and the smallest average litter size was 4. There was extremely significant difference in average litter size between intermediate type and the other two types （P=0.002 1<0.01）， indicating that the intermediate type pigs are more stable in reproductive performance than the other two types of Enshi Black pig.
Average daily weight gain per litter
As shown in Fig. 3， the average daily weight gain per litter of intermediate type was very significantly higher than that of the other two types （P=0.002 2<0.01）.
Comparison of immune traits among Enshi Black pig populations
Analysis of asymmetric data revealed that the total number of white blood cells （WBCs） of intermediate type showed no significant difference from that of Shizitou type （Table 1）， but was very significantly higher than that of Goutou type. The percentage of lymphocytes of intermediate type was significantly higher than that of Shizitou type， and very significantly higher than that of Goutou type. The number of neutrophils of intermediate type was significantly higher than that of the other two types. There was no significant difference in the number of red blood cells and the number of platelets between the three populations of Enshi Black pig.
As can be seen from Table 2， the percentage of CD3+CD4+ T cells， the percentage of CD3+CD8+ T cells and CD4+/CD8+ ratio of the intermediate type were all significantly higher than those of the other two types， indicating that the intermediate type Enshi Black pig has higher immunological activity than the other two types. Correlation analysis on the traits of the three Enshi Black pig populations showed that there was an extremely significant positive correlation between health score and litter size （P<0.01）， an extremely significant negative correlation between litter size and CD4+/CD8+ ratio （P<0.01）. There was a significant negative correlation between litter weight and the number of red blood cells， the percentage of CD3+CD4+ T cells and the number of neutrophils， the percentage of CD3+CD8+ T cells and CD4+/CD8+ ratio， the number of white blood cells and the percentage of lymphocytes. The percentage of lymphocytes and the number of neutrophils （P<0.05）; the percentage of CD3+CD8+ T cells was significantly positively correlated with the number of platelets， the number of white blood cells and the number of neutrophils （P<0.05）. The results further proved that the health， reproduction and immune traits of the intermediate type Enshi Black pig are all better than those of the other two types.
Conclusions and Discussion
In our experiment， the health status of three Enshi Black pig populations during pregnancy was scored by referring to previous study[6]， and the results showed that the health score of the intermediate type pigs stabilized at above 8， while that of the other two types fluctuated noticeably， and the lowest score was 5， indicating that there was an extremely significant difference in the health status between intermediate type and the other two types of Enshi Black pig （P=0.001 4<0.01）. It can be concluded that the health status of the intermediate type pigs is more stable than the other two Enshi Black pig populations during pregnancy， which proves that the intermediate type has higher disease resistance than the other two types.
Deng et al.[4] evaluated the reproductive performance of different Enshi Black pig populations， and found that the litter size of largesized sows （12.26±2.68 piglets per litter） was significantly higher than that of smallsized sows （only 8.62±1.50 piglets per litter）. The study of Huang et al.[7] showed that average litter was 9.96 for Yorkshire sows， 9.52 for Changbai sows and 9.13 for Duroc sow， showing no significant difference among them （P>0.05）. Fu et al.[8] reported that the average litter size of Changbai， Yorkshire and Duroc sows was 10.07， 9.34 and 10.06， respectively， and the number of piglets born alive per litter was 9.26， 8.87 and 9.85. Our results showed that the average litter size of the intermediate type pigs fluctuated slightly at about 10， while that of the other two types fluctuated greatly， with the smallest litter size at only four. There was an extremely significant difference in average litter size among the three Enshi Black pig populations （P=0.002 1<0.01）. These results revealed that the intermediate type of Enshi Black pig has more stable reproductive performance than the other two types. Peng et al.[5] reported that Enshi Black pig grows slowly and reaches a body weight of about 95 kg at nine months of age. The study of Zhang[9] showed that Large White pig has an average birth weight of 1.5 kg， and reaches a body weight of 6.86 kg at 21 days of age. There are also some studies showing that the weaning weight of piglets can be increased to some extent by changing the ambient temperature， the diet of pregnant sows and suckling piglets[10-11]. Our results showed that the average weaning weight of the intermediate type Enshi Black piglets at 28 days of age was 6.27 kg， which was very significantly higher than that of Goutou and Shizitou Enshi Black piglets （P=0.002 2<0.01）， indicating that the intermediate type Enshi Black pig has stable reproductive and growth performance， which may also prove that the intermediate type sows produce much more milk than the other two types under the same feeding condition. In conclusion， the intermediate type Enshi Black pig has better growth traits than the other two types under the same feeding condition.
White blood cells （WBCs） including lymphocytes and phagocytes， are the cells of the immune system that fight disease by making antibodies or engulfing germs[12， 13]. Lymphocytes are directly involved in both antibodymediated and cellmediated immune responses[14]. As nonspecific immune cells， neutrophils can engulf senescent cells and pathogenic microorganisms， and account for a large proportion （65%-75%） of blood WBCs[15]. When inflammation occurs， neutrophils are attracted to the site of inflammation by chemotactic substances released from damaged tissues， release large amounts of lysosomal enzymes， engulf and digest the microorganisms and tissue debris， preventing the spread of pathogenic microorganisms in the body[16-22]. Our results showed that intermediate type had an extremely significant difference from Goutou type， but no significant difference from Shizitou in the number of WBCs. The percentage of lymphocytes of intermediate type was very significantly higher than that of the other two types. The number of neutrophils of lymphocytes of intermediate type was very significantly higher than that of Goutou type， but showed no significant statistical difference from that of Shizitou， suggesting that the main immune traits of the intermediate type Enshi Black pig may be primarily inherited from Shizitou type， and is better than those of Goutou type. To sum up， the immune traits of intermediate type Enshi Black pig are better than those of Goutou and Shizitou types， which can be explained by heterosis between the two breeds. The size and ratio of T cell subpopulations are important indices of the bodys immune function， and the changes in the population and function of T cell subsets will lead to the disorders in the immune system， and subsequent pathological changes[23]. There are two major classes of surface markers of T lymphocytes： one is CD3 marker， which is shared by all T lymphocytes， the other class is subpopulationspecific： CD4 is expressed in helper/inducer T cells， and CD8 is present on suppressor/cytotoxic Tcells. Elevated CD4+/CD8+ Tcell ratio indicates increased ratio of helper T cells and enhanced immune response[24-27]. By analyzing the CD3+CD4+ and CD4+CD25+ lymphocyte subsets of pigs with low immune response to fever vaccine， Wang et al.[28] found that decreased population of CD3+CD4+ lymphocyte subset and increased population of CD4+CD25+ lymphocyte subset in peripheral blood may result in a low immune response to fever vaccine. After conducting a series of studies on the effects of porcine epidemic diarrhea virus on the immune function of dendritic cells， Gao[29] found that the population of CD3+， CD4+ and CD8+ T cells in the lamina propria and villi of the small intestine significantly increased in the piglets immunized with inactivated porcine epidemic diarrhea virus （PEDV） vaccine and with inactivated K88 48 h later. In the present study， we analyzed the lymphocyte subsets of three Enshi Black pig populations， and found that the percentage of CD3+CD4+ T cells， the percentage of CD3+CD8+ T cells， and CD4+/CD8+ ratio of the intermediate type Enshi Black pig were all significantly higher than those of the other two types， which further indicates that the specific immune system of the intermediate type Enshi Black pig is increased compared to the other two types.
Spearman rank correlation analysis showed that there is a significant positive correlation between health score and litter size， the percentage of CD3+CD8+ T cells and the number of platelets. In addition， we also found that there is an extremely significant negative correlation between litter size and CD4+/CD8+ ratio. There is a negative correlation between the number of red blood cells and the weight gain of weaned piglets. There are many other factors influencing the immunity of pregnant sows， which need to be studied in future work. In summary， the intermediate type inherits the excellent traits of both Goutou and Shizitou types， and thus has better growth， reproduction and immune traits. The results may provide a theoretical basis for the detection and identification of genetic variation and conservation of local pig breeds in China. References
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