Study on the mortality pattern of African Swine Fever in pigs during an outbreak in Ranchi, Jharkhand Gupta M.K.*, Kumar Sanjit1, Lakra P.P.1, Senthilkumar1, Rajukumar K.1, Kumar Brajesh1, Pamia Jasmine1, Kumar Ravindra2, Kumar Ajay3, Mahtha B.B.4, Prasad Sushil5 Department of Veterinary Pathology, College of Veterinary Science and Birsa A.H. Agricultural University, Ranchi 1ICAR-NISHAD, Bhopal, MP 2Department of Livestock Production and Management, Government of Jharkhand 3Department of Animal Husbandry, Government of Jharkhand 4Department of Animal Husbandry, Government of Jharkhand 5Department of Livestock Production and Management, Government of Jharkhand *Address for Correspondence Gupta M.K. Professor & Chairman, Department of Veterinary Pathology, College of Veterinary Science and Birsa A.H. Agricultural University, Ranchi, India, E-mail: madhurendu.gupta@gmail.com
Online Published on 27 March, 2024. Abstract The present study on African Swine Fever (ASF) in Ranchi, Jharkhand demonstrates the mortality pattern in different age groups of pigs during an outbreak in 2022. The pigs were also examined for clinical signs before death and, gross and histopa-thology after death during months of August to September were analysed, and the mortality pattern was correlated with the age of pigs at 15 days interval. Among different age groups, the mortality in the starter (0-40 days) age group was lower (19.82%) during the initial phase of the outbreak compared to grower (41-120 days) and adult-finisher (>121 days) which was 35.15% and 78.54% respectively on 15th day. At 45th day, the mortality reached 100% in the adult finisher group whereas it was 72.69% and 82.53% for starter and grower groups respectively. The grower and starter lot showed 93.23% and 92.17% mortality respectively by the end of the outbreak (60th day) season. A comparative analysis of the overall mortality among different breeds showed a non-significant difference (p< 0.05). At necropsy, the prominent gross lesions were splenomegaly, haemorrhagic hepatic and mesenteric lymph nodes, epicardial and endocardial haemorrhages, haemorrhagic gastroenteritis, interstitial pneumonia, multifocal hepatic necrosis, and congested kidneys in most of the animals, where as renal petechial haemorrhages was observed in one pig which died later during the outbreak when mortality had subsided. Histopathologically, the lesions were renal tubular necrosis, hepatic sinusoidal congestion and vacuolar degeneration, massive congestion and haemorrhages in cortical and medullary areas of spleen and lymph node, heart showed haemorrhage and congestion with pyknotic changes in the cardiomyocytes. Top Keywords African swine fever, Age, Breed, Gross pathology, Histopathology, Mortality pattern. Top |
Introduction African Swine Fever (ASF) is a highly contagious disease of both farm raised as well as wild pigs. It was first detected in Kenya in the year 19211 from where it spread to all Sub-Saharan African countries and later in the year 1957, reported in Portugal and other European, North and South American countries2. Subsequently, the disease rapidly spread to Asian countries like China, Indonesia and Myanmar by 2018. In India, the disease was first recorded from Assam3 and Arunachal Pradesh in 2020 and the possible source of ASFV was suggested to be from China4. ASF is classified as a notifiable disease by the World Organization for Animal Health (OIE, 2008). |
Pig farming is a significant source of livelihood in Jharkhand and plays an important role in the rural economy, especially in the tribal belt. Pig farming has gained considerable popularity in the state. Most of the tribal people rear pigs in the backyard along with other livestock and poultry. Pigs also thrive well in the agro climatic conditions of the state and its meat serves as an important source of protein in the rural Jharkhand. The total pig population of Jharkhand as of 2019 census was 12.8 lakh. In 2022, suspicious death of pigs were reported in large numbers from all over Jharkhand from the month of July onwards which was later confirmed to be due to ASF by National Institute of High Security Animal Diseases (NIHSAD), Bhopal, India. The present work was taken up to study the age and breed wise mortality pattern and pathology of ASF in pigs of Ranchi and surrounding areas. |
Top Materials and Methods The study was conducted on pig mortality recorded in the Government Pig Farm (GPF) managed by Department of Animal Husbandry, Government of Jharkhand, located in Kanke area of Ranchi district, and in the Instructional Pig Farm (IPF) of College of Veterinary Sciences and Animal Husbandry, Kanke, Ranchi. Both the farms are located at a distance of 500 metres. The GPF, Kanke and IPF, CVSc & AH, Kanke, Ranchi housed 2034 and 1575 pigs of different age groups respectively at the start of the study. The following parameters were examined during the present study. |
Clinical signs The clinical signs exhibited by the animals of different age groups suffering from ASF in both the pig farms under study were critically observed and recorded from the onset of disease outbreak and during its progression. Gross pathology Post mortem examination was conducted on six animals in early phase of mortality in GPF and two on each day during the first three days of outbreak in IPF, Ranchi. During post-mortem examination, every organ was examined critically and the gross lesions present in different organs of dead pigs were recorded. Once the disease was confirmed, the post-mortem examination was stopped at both the places. Histopathology Small pieces of tissue were collected from spleen, lymph nodes, heart, stomach, intestine, lungs, liver, and kidney during necropsy and preserved in 10% buffered formalin for histopathological examination. The tissues were processed for histopathology as per the standard technique of Bancroft and Gamble5. The sections were stained with Haematoxylin and Eosin stain and DPX mounted permanent sections were critically examined for histopathological alterations in different organs of the body. Confirmation of the disease Samples of tissue from spleen, lymph nodes, heart, stomach, intestine, lungs, liver and kidney were collected during PM examination and preserved in transport media and in 10% neutral buffered formalin along with 18 whole blood samples in EDTA. The serum samples were submitted to Director ICAR-NIHSAD, Bhopal, for confirmation of the disease causing pathogen through molecular diagnostic techniques such as RT-PCR and nucleotide sequencing. Mortality pattern Mortality pattern in starter (0-40 days), grower (40120 days) and adult animals (more than 120 days) of both the farms were studied at 15, 30, 45 and 60 days of the onset of clinical ASF. Statistical analysis The data generated during the study period was subjected to Chi-Square test as given by Snedecor and Cochran6 to understand the statistical significance of variation in the mortality pattern amongst different breeds and age group of pigs. Top Results It was observed that pigs reared in GPF suffered from heavy mortality at the rate of 10-15 pigs/day starting from last week of July 2022, and continued till August 2022. Overall 98.38% mortality was recorded during the outbreak, resulting into survival of 33 out of 2034 pigs. The outbreak of similar nature in the adjacent IPF started in the last week of August 2022 i.e. about one month after the initial outbreak and continued till the end of September 2022. In the extended outbreak 98.35% mortality was recorded with survival of only 20 pigs out of 1575 at the end. |
Clinical signs The diseased pigs in both the farms exhibited similar clinical signs like high fever (104-106OF), depression, anorexia, bleeding from natural orifices (Fig. 1), highly congested and cyanotic skin particularly in the ventral abdomen and neck region. Death usually resulted within 24 hours of the onset of clinical signs. The disease initially affected the animals of one pen; however, later it spread to other pens. Morbidity and mortality was higher in adult animals initially, but later the disease was found to affect growers and starters too. No difference was observed in the clinical signs of the disease between different age groups and breeds of pig. Gross Pathology The most common organs affected during the outbreak of ASF in decreasing order of frequency were spleen, lymphnode, epicardium and endocardium of heart, lungs, stomach, small and large intestine, liver and kidney. The major consistent gross pathological lesions in the pigs of both the farms were splenomegaly, where spleen showed more than two to three times enlargement in size as compared to normal (Fig. 2). Lungs manifested bilateral pneumonia with patches of haemorrhages on its surface. Heart consistently showed marked epicardial and endocardial haemorrhage in almost all the animals died due to ASF (Fig. 3), with ventricles mostly found devoid of blood. The mesenteric and hepatic lymph nodes showed marked haemorrhagic and oedematous changes in all the animals (Fig. 4). Gastrointestinal tract revealed marked haemorrhagic gastroenteritis with development of large patch of dark red haemorrhages in the fundic mucosa of greater curvature of stomach, Intestine mainly revealed widespread petechial and linear mucosal haemorrhages all along its length (Figs. 5 and 6). None of the dead animals exhibited button ulcer in the intestinal mucosa. Liver characteristically showed multifocal hepatic necrosis with round, pale, shallow necrotic lesions measuring 3-5 mm in diameter (Fig. 4). Although both the kidneys in affected animals showed congestion, however petechiations or turkey egg appearance was not a feature in any of the affected animals except one (Fig. 7) which was recorded when death took place after acute phase of outbreak had passed. The blood examination of this animal was found to be negative for ASF. Histopathology Characteristic microscopic lesions were observed in visceral organ of pigs from both the farms. Spleen revealed significant increase in red pulp (Fig. 8), sub-capsular oedema and marked infiltration of mononuclear cells in the sinuses. Lymphnode showed haemorrhages in both cortical and medullary area with islands of lymphoid follicles giving characteristic washed out appearance. There was sub-capsular oedema and highly congested blood vessels (Fig. 9). Lungs revealed congestion and haemorrhage with interstitial pneumonia characterised by predominant infiltration of mononuclear cells along with few neutrophils in the interstitial spaces. Heart showed congestion and haemorrhage with pyknotic changes in the cardiocytes. Congestion and haemorrhage in submucosa and muscularis mucosa along with degeneration of glandular tissue at few places was also observed. Stomach revealed mononuclear cell infiltration in pyloric glands, highly congested large blood vessels, as well as haemorrhagic and degenerative changes in the gastric mucosa. Intestine showed haemorrhage, oedema and marked degeneration of villi (Fig. 10), as well as inflammatory cell infiltration in lamina propria and submucosa. The inflammatory cells predominantly consisted of lymphocytes and macrophages. Liver showed highly congested sinusoids, coagulative necrosis and vacuolar degeneration of hepatocytes (Fig. 11), proliferation of bile ducts, and formation of hyaline bodies of variable size in the hepatocytes. Hepatocytes also showed pyknotic and karyorrhectic cellular changes. Moderate degree of mononuclear cell and eosinophil infiltration was observed in the portal triad. Kidney showed marked coagulative necrosis of tubular epithelium characterised by cytoplasmic acidophilia, and pyknotic nuclei (Fig. 12). At places there was complete detachment of tubular epithelium with their aggregation in the lumen. Glomerular tuft revealed hyperplastic changes. Mortality pattern The disease exhibited over 98% morbidity and mortality in the pigs of both the farm. The age wise and breed wise details of the mortality pattern in both the farms are presented in Figs. 13 and 14, respectively. Variable mortality percentage was observed amongst different age group of pigs irrespective of breeds. Mortality percentage in adult pig reached 78.54% (015 days) and 95.77% (15-30 days) by the end of first month. Moderate mortality (72.05) was observed in grower while least mortality (43.47%) was recorded in the starters during first month of outbreak. However in the second month 100% mortality was observed in adult pigs by 45th day, whereas in grower and starter pigs, mortality reached 93.2 and 92.17% by the end of 60th day (Table 1, Fig. 13). Survival was seen only in grower and starter pigs during the current outbreak. Thus, higher mortality was recorded in adult animals from the very beginning in both the farms. Chi-square analysis revealed that there was significant age wise difference in the mortality percentage in first 30 days of the outbreak (Table 2). Survival percentage of pig was found to be 1.62% and 1.65% in the GPF and IPF respectively. Breed wise variation was observed in the mortality percentage which revealed highest mortality in Ghungroo (100%) followed by Large White Yorkshire, Jharsuk, Tamworth and Hampshire. Least mortality (69.44) was observed in Russian Charmukha breed of pig (Table 3, Fig. 14). However, the difference in mortality percentage between different breeds of pig was found to be statistically nonsignificant (Table 4). Confirmation of the disease Analysis of whole blood, serum and tissue samples was carried out at ICAR- NIHSAD, Bhopal, India by RT-PCR followed by nucleotide sequencing of the isolated virus particle isolated. Investigation of eleven samples submitted by GPF, Kanke, revealed that 4 nasal swab, 3 rectal swab, one clotted blood and three tissue samples were found positive for ASF while one sample of clotted blood was found negative for ASF. At the peak of the outbreak the positivity percentage was found to be 91.1% in whole blood, serum and tissue samples. In the subsequent outbreak at IPF, five whole blood samples, three from live and two from dead animals and five serum samples, three from live and two from dead were submitted for investigation at ICAR-NIHSAD, Bhopal, India. Test result revealed that positivity percentage in both serum and whole blood samples from dead animals was 100% while serum and whole blood samples from live animals during the peak of ASF outbreak was only 33.3%. The random samples collected after one and half month of initial outbreak from one Hampshire, one Tamworth, four Jharsuk, three LWY and one Russian Charmukha pigs of IPF were found negative for ASF. The tissue samples of pig which apparently showed classical turkey egg appearance of kidney was also found to be negative for ASF. Top Discussion Jharkhand is a major pig farming state of India. It boasts a pig population of 12.8 million which is only second to Assam as per 2019 livestock census. As the state is in close proximity to north-eastern states, it is always susceptible to interstate crossover of disease. The etiological agent of ASF is African swine fever virus (ASFV), a member of family Asfarviridae, and Asfivirus varies in length between 170-193 kbp7. The occurrence of ASF in India was first reported from adjoining states of Arunachal Pradesh and Assam in 2020 as trans-boundary disease when virus crossed over from China4. |
The virus with its adaptability to replicate in its hosts in the sylvatic cycle, poses special threat to the pig population of Jharkhand state due to its vast forest area and presence of wild boar susceptible to ASFV, thus increasing the possibility of recurrence of the disease in future as they also maintain the virus. Its capability to spread rapidly to different regions through wild life hosts, climatic conditions, pig management system and blatant disregard for biosecurity measures in rural population makes it a very dangerous pathogens for pig farming industry of Jharkhand and India. The entry of virus into the body cause viraemia in 3 days' followed by its presence in nasal and rectal fluid by 10-13 days' can subsequently infect the pen mates through inhalation or ingestion of infected material7. ASFV is usually transmitted between infected pigs by direct contact through oral-nasal route or through skin abrasions. Bleeding from natural orifices was a common clinical manifestation in the present outbreak, hence rapid spread within both the pig farms must have been through direct or indirect contact of pen mates within infected premises, through ingestion of infected material or indirectly through attendant. However, spread of virus from GPF, to IPF, located 500 meters apart with no direct connection must have been through aerosol route because virus can travel short distance through aerosol route8, 9. The outbreak in Jharkhand was controlled by imposing strict ban on the movement of pigs in the state and in the locality subsequent to confirmation of the disease by ICAR-NISHAD, Bhopal, India. Massive awareness drive was taken up with establishment of protection and surveillance zone in an area of 3 km and 10 km, respectively. |
Major gross lesion observed during the outbreak comprised of marked splenomegaly and lymphadenitis of hepatic and mesenteric lymphnodes with haemorrhagic and oedematous changes. Haemorrhages and congestive changes were also recorded in epicardium, endocardium, gastric mucosa, intestinal mucosa and lungs etc. The present findings are similar to the observation of10. Haemorrhagic and hyperaemic splenomegaly is considered as most typical lesion of ASF and the severity of lesion depends on virulence of the isolate11, 12. |
Histopathologically, marked haemorrhagic and necrotic changes were observed in the spleen, lymphnodes, endocardium, epicardium, as well as in gastric and intestinal mucosa. Similar histopathological findings in ASF outbreak has been reported earlier10, 13. Washed out appearance of lymphoid follicles was a major consistent change in lymph nodes and spleen, which makes the animal immunodeficient. Lymphoid cell depletion in the lymphoid organs during ASF has been recorded by Gomez-Villamandos1. Increased TNF-a, IL-1a, IL-1|3 and IL-6 cytokine expression has been reported in lymphoid organs along with increased serum level of TNF-a and IL-1|3. These changes bring about apoptotic loss of lymphocytes as well as create cytokine storm with resultant disseminated intravascular coagulopathy (DIC) and consumption coagulopathy getting expressed as widespread petechial, ecchymotic and haemorrhagic lesions and accumulation of transudate in the body cavity10. TNFa is reported to play a key role in the pathology of ASF by inducing alteration in vascular permeability, coagulation and induction of apoptosis in uninfected lymphocytic population14. |
Higher depletion of cells in the lymphoid follicles is suggestive of higher virulence of the virus,15, 16 this confirms that highly virulent strain of ASFV prevailed in the state of Jharkhand. Mononuclear cell predominantly macrophage infiltration was commonly seen histopathologically in different organs. Lymphopaenia and lymphoid depletion in lymphoid organ during ASF has also been described13. The cause of cellular damage observed in lymphoid and other organs has been attributed to activation of macrophage triggered cytokine mediated interaction rather than virus induced17. |
The phagocytic activation of capillary endothelial cells with resultant hypertrophied endothelial cells culminates in obliteration of the capillary lumen, leading to increased intravascular pressure18, loss of endothelial cells and exposure of the capillary basement membrane with resultant microthrombus formation, petechiations, ecchymosis and exudation of oedema fluid18, 19. |
The liver in pig suffering from ASF mostly exhibited marked congestion and also multifocal periportal inflammatory cell infiltration, similar findings have been reported earlier10,13,20-23. These lesions are responsible for mottled appearance of liver. In the present study, prominent multifocal hepatic necrotic lesion was a consistent feature which has not been described by earlier workers. It might be due to highly virulent nature of the virus substantiated by the fact that haemorrhagic lesions too were quite extensive in heart, spleen, entero-hepatic lymph nodes, gastric mucosa and intestinal mucosa. |
Reduced incidence of ASF in starter and grower pigs in the initial one month of outbreak could be attributed to less production of pro inflammatory TNF cytokines including Leukotriene A, Leukotriene B and Fas Lig and probably due to less developed macrophage-monocyte system in the early life. However with age it reaches optimal functioning which might induce massive cytokine storm and heavy mortality in animals24. Contrary to our observations, higher resistance to ASFV in older pigs as compared to younger pigs has been described in pig breeds25. |
The mortality pattern in different breeds and varieties of pigs showed relative resistance to ASFV in Russian Charmukha, Hampshire, Tamworth and Jharsuk. Large White Yorkshire was found to be most susceptible. However, the variation in mortality rate between different breed and varieties of pig were non-significant. The gene associated with resistance to ASFV infection is termed as RELA (Reticuloendotheliosis viral oncogene homolog)26. The gene causes the immune system to over react as it detects the disease. Significant variation in the expression of RELA genes between resistant and susceptible pig is associated with host response to ASF infection. Another cause of resistance may be innate immune system and its ability to control viral replication resulting in reduced systemic infection. This might be the cause of variable susceptibility of different varieties of pigs. There is uncertainty regarding genome connected with virulence of ASFV and needs further exploration. The two ASFV virulence genes are UK and 23-NL located adjacent to each other in right variable region of the genome. Deletion of UK and 23-NL genes diminishes the virulence of ASFV without affecting virus replication in macrophages27, 28. Previous findings suggest that genotype II isolates of the "Georgia 2007 type" strain prevalent in eastern and central Europe and recently in Asia are highly virulent and cause mortality in the range of 91-100%29. The nature of virus involved in Ranchi outbreak needs further exploration on above line of action. |
In the Ranchi (Jharkhand) outbreak it has been observed that 100% tissues collected from dead animals were positive for ASF, whereas the positive percentage was only 33.3% when the samples from live animals during the outbreak were tested for ASF by molecular diagnostic technique. This makes the tissue collected from dead animals a better sample for diagnosis of ASF through molecular techniques in future studies. Another interesting finding was that the investigation done for diagnosis of ASF from surviving animals after one and half month of outbreak were all negative for ASF. This is contrary to the report of30 which mentioned that survivors may develop persistent infection in susceptible tissues of the body and cause disease under favourable conditions such as immunosupression, transportation or irregular and under feeding to the animals. |
Thus, it can be concluded that the lower age group showed significantly lower mortality than the other two age groups during the outbreak. More research is required to understand some of the pathogenetic mechanisms, including how ASFV modulates the host immune responses in different age groups which make them less susceptible to ASF. This appears to be the first report of ASF in Jharkhand. |
Top Figures | Fig. 1:: ASF infected pig showing bleeding from natural orifices
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| | Fig. 2.: Heart showing endocardial haemorrhage on both medial ventricular walls.
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| | Fig. 3:: Spleen showing marked enlargement and congestion
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| | Fig. 4.: Liver showing multifocal necrotic lesions along with severely swollen and haemorrhagic hepatic lymphnodes
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| | Fig. 5.: Stomach showing severe haemorrhagic gastritis with fibrinous exudates
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| | Fig. 6.: Intestinal mucosa showing petechial and ecchymotic haemorrhages
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| | Fig. 7.: Kidney showing Petechial haemorrhage (Turkey egg appearance).
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| | Fig. 8:: Spleen showing marked increase in the red pulp due to haemorrhage (H&E x100)
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| | Fig. 9.: Lymphnode showing washed out appearance of lymphoid follicles in ASF (H&E x100)
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| | Fig. 10.: Intestine showing mucosal degeneration with loss of villi, and infiltration of inflammatory cells in the lamina propria and submucosa (H&E x100).
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| | Fig. 11:: Hepatocytes showing coagulative necrosis in periportal area, as well as portal hepatitis (H&E x100)
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| | Fig. 12.: Kidney showing coagulation necrosis of tubular epithelial cells (H&E x100).
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| | Fig. 13:: Mortality trend in pigs of different age group at 15 days interval (Graph).
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| | Fig. 14:: Mortality percent of different breeds during the outbreak of ASF (Graph).
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Tables | Table 1:: Mortality percent in pigs of different age group at 15 days interval.
| | Days | Adult - Finisher | Grower | Starter | | 0-15 | 78.54% | 35.15% | 19.82% | | 16-30 | 95.77% | 72.05% | 43.47% | | 31-45 | 100% | 82.53% | 72.69% | | 46-60 | 100% | 93.23% | 92.17% |
| | | Table 2:: Chi-Square analysis of the mortality between different age groups at 15 days interval.
| | Days | Between groups | P value | | 0-15 days | Starter x Grower | 17.588** | | Starter x Adult-Finisher | 115.883** | | Grower x Adult-Finisher | 43.097** | | 16-30 days | Starter x Grower | 23.449** | | Starter x Adult-Finisher | 53.128** | | Grower x Adult-Finisher | 7.122** | | 31-45 days | Starter x Grower | 10.019** | | Starter x Adult-Finisher | 1.798NS (p<0.05) | | Grower x Adult-Finisher | 3.392NS | | 45-60 days | Starter x Grower | 0.016NS | | Starter x Adult-Finisher | 0.687NS | | Grower x Adult-Finisher | 0.464NS |
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| **Significant at p<0.01; *Significant at p<0.05; NS - Non-significant | | | Table 3:: Mortality percent of different breeds during the outbreak of ASF.
| | S.No. | Breed | Mortality percent | | 1. | LWY | 93.69% | | 2. | Tamworth | 87.50% | | 3. | Ghunghroo | 100% | | 4. | RC | 69.44% | | 5. | Hampshire | 70.37% | | 6. | Jharsuk | 92.59% |
| | | Table 4:: Chi-Square analysis of overall mortality in different breeds during the outbreak of ASF.
| | S.No. | Between groups | P value | | 1. | LWY x Tamworth | 0.030NS (p<0.05) | | 2. | LWY x Ghunghroo | 0.017NS | | 3. | LWY x Russian Charmukha | 1.042NS | | 4. | LWY x Hampshire | 1.294NS | | 5. | Tamworth x Ghunghroo | 0.050NS | | 6. | Tamworth x Russian Charmukha | 0.265NS | | 7. | Tamworth x Hampshire | 0.266NS | | 8. | Ghunghroo x Russian Charmukha | 0.460NS | | 9. | Ghunghroo x Hampshire | 0.465NS | | 10. | Russian Charmukha x Hampshire | 0.0016NS |
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| **Significant at p< 0.01; *Significant at p<0.05; NS - Non-significant | |
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