Evaluation of Crossbreeding Parameters for Immunocompetence and Serum Enzyme Profile in a Partial Diallel Cross Involving Three Genetic Groups of Chicken

Over the past few decades, the poultry sector has registered phenomenal growth in terms of volume of production and individual productivity. The scientific and technological advances, largely applied in organised poultry ventures only could bring poultry to present status of production. The unorganized sector primarily dominated by rural poultry production is largely a non-intensive venture, predominantly comprising indigenous chicken with relatively lower production potential. Native chicken breeds or their crosses are capable of thriving well in tropical climate owing to their adaptability, disease resistance and inherent resilience (Thapa, 2018). By adopting breeding strategies with indigenous germplasm, the new dual-purpose crosses with augmented production potential and immunocompetence for production in low inputs system, are required to be developed through appropriate breeding strategy.
       
The diallel/partial diallel mating designs have been extensively used for identifying the best nicking lines or breeds for achieving higher heterotic effects in the crosses (Gupta et al., 1995 and Nath et al., 2007) as well as for assessing the genetic variability and combining abilities of complexly inherited economic traits (Rajkumar et al., 2011).
       
The partial diallel cross is a refitted version of diallel cross wherein all purebreds and few selected crossbreds are produced after exclusive selection of distinct male and/or female lines. It is one of the pragmatic tools in computing the combining abilities and maximizing the genetic gain through identification of the best parent lines and cross combinations. Besides, curtailing the number of crosses reduces the resource requirements and breeding design become less cumbersome. In the present study, the partial diallel experiment was designed comprising three chicken breeds/genetic groups viz. coloured synthetic male line (CSML), Local Desi or native chicken (D) and one exotic chicken germplasm- CARI-Red (CR) for producing the pure and crossbred progenies for evaluating their performance and crossbreeding parameters for immunocompetence traits and serum enzymes with an ultimate objective to identify the best combining male and female lines for producing the cross having better disease resistance and adaptability to the rural conditions.

The present study was conducted at the Experimental Broiler Farm, ICAR-Central Avian Research Institute, Izatnagar, Bareilly, (UP) following approval from Institute’s Animal Ethics Committee.
 
Experimental flock
 
In this study, the germplasms used as parent stock were-1) Colored Synthetic Male Line (CSML)- a colored broiler parent line developed at the institute’s broiler farm from synthetic base population, 2) S3 generation of native chicken (Desi) and 3) CARI-Red (CR)- an exotic germplasm developed by adapting and pure breeding the Dahlem Red breed of chicken maintained at CARI, Izatnagar. Besides, the females of cross CSML×CR developed in a previous study (Thapa, 2018) at CARI were used to produce a triple cross.
 
Mating plan: Partial diallel cross
 
Genetic group wise sires and dams were used in the partial diallel cross (Table 1) in mating ratio of 1:3 to produce pure and crossbred progenies by artificial insemination. Additionally, a triple cross was developed by using the sires of Local Desi chicken and dams of (CSML×CR) cross developed earlier by Thapa (2018).
 

 
Managemental practices
 
From day one, all the standard managemental practices including biosecurity and vaccination were followed. The chicks were maintained on deep litter system up to 20 weeks of age and were provided with ad lib standard soya-maize based starter (up to 3 wk) and grower ration as per the ICAR feeding standard (ICAR, 2013) there onwards up to 20 weeks of age.
 
Evaluation of immunocompetence
 
Humoral immune response (HIR)
 
Blood was collected aseptically from 12 birds per group at 21 days post primary vaccination for ND and the serum was harvested. The hemagglutination inhibition (HI) test was done by the procedure of King, (1999) with two-fold serial dilution of serum to assess HIR as antibody titre against ND vaccine. The maximum dilution showing HI (central button at bottom of the well) was recorded as the end dilution with positive HI titre to express as its Log₂ value.
 
In-vivo cell-mediated immune response (CMIR)
 
The CMIR was studied by the method of (Corrier and Deloach, 1990) at six weeks of age by intra-dermal injection of 0.1 ml (1mg/ml) of phytohemagglutinin type-P (PHA-P) at 3^rd and 4^th inter-digital space of the right foot and 0.1 ml of sterile PBS in the left foot (control). The foot web index (mm) was calculated as the difference between the swelling in the right and left foot before (R1 and L1) and after (R2 and L2) 24 hours of injection. The formula used for the calculation of CMIR was- CMIR= (R2-R1)-(L2-L1).
 
Relative weight of immune organs
 
At 8 and 12 weeks of age, eight chickens per group were weighed and euthanized by a standard method of slitting the throat and exsanguinations, followed by careful separation, wiping and weighment of spleen, bursa of fabricius and thymus individually. Weights of immune organs were expressed as percentage of pre-slaughter live weight.
 
Serum enzymes levels
 
At the 8^th and 12^th week of experiment, serum was harvested from eight birds per group and levels of enzymes viz. aspartate transaminase (AST), alanine transaminase (ALT) (Modified method IFCC, 1986) and alkaline phosphatase (method of Kind and King, 1954) were assessed using standard kits from M/s Coral Clinical System Diagnostic Ltd, Goa, India.
 
Statistical analysis
 
The data were analysed using one-way ANOVA by SPSS-20 software as per the methods of Snedecor and Cochran (1980). Subclass means were compared using Tukey’s post- hoc test (Tukey, 1953).
 
Diallel analysis
 
The general and specific combining ability effects and other crossbreeding parameters were computed as per model-II of method-II by Griffing (Griffing, 1956).
 
Determination of heterosis
 
The percentage of heterosis (H), heterobeltiosis (Hbt) and standard heterosis (Hst) in crossbred progenies were computed by using the following formula.

Immunocompetence traits
 
CMI and HIR differed significantly (p<0.01) among different genetic groups (Table 2). CR×CR exhibited superior foot web index and HI titer against NDV among purebreds followed by D×D and CSML×CSML. Among crossbreds, D×CR showed highest CMIR as well as HI titre, followed by triple cross and CML×D. Overall, CR×CR elicited better immune response followed by D×D and CSML×CSML among purebreds. Whilst in crossbreds, D×CR and triple were superior. Reports on investigating CMI (Shivakumar and Kumar, 2005; Sivaraman et al., 2005) and HIR (Cheema et al., 2003; Reddy et al., 2002) in various genetic groups of chicken are widely available. In an investigation similar to present study, Thapa, (2018) reported significantly higher CMI and HIR in D×D followed by CR×CR and CSML×CSML in purebreds, whereas in crossbreds CSML×D revealed better immunity followed by D×CR and CSML×CR.
 

       
Haunshi and Sharma (2002) found significantly higher HA titre in Dahlem Red than Aseel, Kadaknath and white leghorn. Van Boven et al., (2008) suggested that, HI titer against NDV should be greater than 2⁸ (1:256) in at least 85% of the flock after two vaccinations to generate herd immunity, nonetheless, HI levels of 1:32 (2⁵) or higher are also thought of being protective (Allan et al., 1978).
       
Ahmed et al., (2007) inferred that the birds with high response to PHA-P have better innate and adaptive immunity to velogenic NDV, while Singh and Singh, (2004) did not find any significant difference for CMI to PHAP among Aseel, Kadaknath, Frizzle and Naked neck. Vander Zijpp et al., (1983) implied that both CMI and HIR be combined to select for general improvement of immunocompetence in chicken due to lack of correlation amidst them.
 
Immune organs weight        
 
At 8 weeks of age, significantly higher (P<0.01) spleen weight was observed in D×CR followed by CSML×CR, whereas lowest was recorded in Local Desi purebred (Table 2). At 12 weeks, CR purebred showed significantly higher (P<0.001) spleen weight followed by CSML×CR while CSML purebred had the least relative spleen weight. The crosses with CR as parents revealed higher spleen weight, which may correspond with comparatively superior CMI and HIR in these crosses. At 8 weeks of age, statistically significant change in weight of bursa could not be observed among different genetic groups. However, at 12 weeks, CR×CR showed significantly higher (P<0.05) bursa weight followed by D×CR. Lowest bursa weight was noticed in CSML×D and CSML×CSML at 12 weeks. Weight of thymus at 8 weeks of age was significantly higher (P<0.05) in D×CR followed by CR×CR and CSML×CR. While at 12 weeks, triple cross had significantly higher (P<0.05) thymus weight followed by D×CR. At both the ages, CSML×CSML had the lowest thymus weight among all the pure and crossbreds.
               
At both 8 and 12 weeks, collective immune organ weight differed significantly (P<0.001). Among the purebreds, CR×CR showed in higher immune organ weight followed by D×D and CSML×CSML. Among the crossbreds, D×CR showed the highest immune organ weight followed by CSML×CR, whereas, CSML×D being the least among all at both the ages.
       
Immune organs deliver a site for maturation and storage of lymphocytes. Therefore, the collective weight of immune organs is a reliable measure of immune response as it offers the delineative picture by neglecting the variations in individual organs. It is well established that the relative weight of immune organs tends to show proportional reduction due to their age dependent atresia and simultaneous body weight gain. This can be correlated with our results. Desi and CARI-Red derived crosses had the superior immune organ weight, thus, exhibited superior immune response in general. Similar results were reported by Thapa, (2018).
 
GCA and SCA for immunocompetence traits
 
Between the genetic groups variances of GCA were non-significant for both, CMI and HIR. CARI-Red showed positive and the highest GCA estimates followed by Desi for CMI whilst, GCA estimates for HIR were equal and the highest in CR and Desi (Table 3). GCA is a magnitude of additive genetic variance for immunity traits which can be improved through selective breeding. Variances for SCA revealed significant difference (P<0.01) for NDV titre, while, CMIR being non-significant. CSML×CR and D×CR showed negative SCA estimates for HIR and CMIR, despite Desi and CR had the highest GCA value. Schilling et al., (1968) suggested similar possibility, where higher GCA value of parents could have low SCA value for the corresponding trait.
 

       
GCA variances for immune organ weight at both the ages were non-significant (Table 3). Desi yielded higher and positive GCA estimates followed by CSML, while CARI-Red being negative at both the ages (Table 3). Variance for SCA differed significantly (p<0.01) only at 12 weeks for immune organs. D×CR showed positive and highest SCA estimates for immune organ weight followed by CSML×D at both ages. Significant SCA variances for NDV titre and organ weight (12 weeks) depict the role of non-additive genetic variation which was also reported earlier by Thapa, (2018). Overall, higher GCA was observed for CMI and HIR in CARI-Red followed by Desi, for as much as immune organ weight, better GCA estimates were exhibited by Desi. CSML×D showed overall higher SCA estimates for immune traits.
 
Heterosis in immunocompetence traits
 
Heterosis (H%), heterobeltiosis (Hbt%) and Standard heterosis (Hst%) for immune response traits and relative immune organ weight of crossbreds are depicted in Fig 1 and 2 respectively. Positive heterosis was observed only for CSML×D and triple cross for CMI and HIR respectively, while all other crosses being negative. For both the traits, Hbt% were negative for all the crosses. Except D×CR, Hst% for both traits were positive for all the crosses, with CSML×D and triple cross having the highest Hst% for CMIR and HIR respectively. For immune organ weight, positive heterosis was recorded in all the crosses at both the ages except triple cross at 8 weeks. Hbt% was positive for (CSML× D and D×CR at 8 weeks and D×CR and triple cross at 12 weeks respectively. Hst% was positive for all crosses at both ages.
 

 

               
In general, negative heterosis and Hbt were obtained for CMI and HIR for crossbreds, indicating superiority of mean immune response of parent breeds than their progenies. However, positive Hst% in almost all crosses (except D×CR for HIR) shows better immune response of crosses than the corresponding inferior parent breed. Nath et al., (2001) concluded the role of non-additive components of variance with positive heterotic effects. Therefore, absence of significant additive and non-additive genetic action in our study for CMI might be responsible for the negative heterotic affects in crossbreds. Haunshi and Sharma, (2002) also could not find consistent sizable heterotic effect on immunocompetence traits.

Serum enzymes
 
Serum enzymes levels at 8 and 12 weeks of age in different genetic groups are given in Fig 3.
 

 
Serum AST and ALT
 
Serum AST levels were significantly higher (P<0.001) in triple cross followed by D×CR and CSML×D cross at 8 weeks of age. The lowest AST levels were recorded in CSML×CSML. On the other hand, at 12 weeks of age, similar trend was noticed, wherein triple cross showed significantly higher (P<0.01) levels followed by CSML×D, while CSML×CSML being the lowest. The serum ALT levels showed results similar to those for AST at 8 weeks, nonetheless, the lowest level was recorded in D×D. At 12 weeks, albeit statistically insignificant, elevated ALT levels in CSML×CSML followed by CSML×D were evident, while D×CR recorded the lowest levels of all.
       
Negative correlations between age and serum AST (r=-0.125) and ALT (r=-0.211) levels were noticed as a general trend in the crosses. Senanayake et al., (2015) reported positive (AST) and negative (ALT) correlation between age and enzymes levels in broilers. AST and ALT are found in highest concentration in liver and their serum levels are used to diagnose hepatotoxicity (Króliczewska et al., 2017), acute liver failure and other stress conditions, as serum transaminase levels exhibit an increase upon hepatocellular damage (Silanikove and Tiomkin, 1992). High metabolic demands in early growth phase tends to elevate liver activity and enzyme synthesis (Senanayake et al., 2015), which might be the cause of elevated AST and ALT levels at 8 weeks of age compared to 12 weeks in our results. However chronic heat stress may cause hepatic insufficiency and reduced enzyme turnover.
 
Serum AST/ALT ratio
 
AST/ALT ratio differed significantly (P<0.01) at 8 weeks of age, but did not exhibit significant change at 12 weeks. D×CR resulted in higher AST/ALT ratio followed by triple cross, while, CSML×CSML exhibited the lowest ratio at both the ages. Positive correlation (r=0.08) between age and AST/ALT ratio was detected. Lowered hepatic enzyme secretion might be a result of adaptive response to adverse conditions and stressors. Also, sudden death syndrome in broilers can be associated with AST/ ALT ratio (Qujeq and Aliakbarpour, 2005). Net values of AST and ALT are comparatively less in CSML, albeit, the age dependent rise in enzyme levels and AST/ALT ratio suggests comparatively poorer resilience to stress, therefore, more likely to encounter with sudden death syndrome.
 
Serum alkaline phosphatase (ALP)
 
The serum alkaline phosphatase levels at 8 weeks were significantly higher (P<0.001) in triple cross followed by CSML×CR and CSML×D, whereas, the lowest levels were recorded in D×CR. At 12 weeks of age, CSML×D yielded significantly higher (P<0.01) ALP levels followed by D×CR, with lowest levels recorded in D×D, depicting slightly different trend. Alkaline phosphatase is extensively present in all the types of tissue and gets activated in alkaline pH owing to hepatic damage and as a by-product of active bone formation (Farley and Baylink, 1986). ALP levels exhibit wide but inconsistent variations due to age, thermotolerance and stress exhibited due to poor husbandry conditions and insufficient vitamin D intake (Senanayake et al., 2015). In the present study, elevated ALP levels at 8 weeks of age may be attributed to high metabolic turnover from bones upon rapid growth. D×CR resulted in lower ALP levels at early age (8 weeks), possibly due to relatively slower growth, yet, the levels were elevated at later stage (12 weeks).
 
GCA and SCA for serum enzymes
 
The variances for GCA and SCA differed significantly for AST, ALT (p<0.001) and ALP (p<0.05) levels at both the ages. CSML showed negative GCA estimates for serum enzymes except for ALT at 12 weeks and ALP at 8 weeks of age (Table 4). Desi recorded positive GCA estimates for AST at both ages, while, ALT estimates were negative at 12 weeks. Estimates for ALP were negative at both ages for Desi. For CR, both AST and ALT showed positive and negative GCA estimates at 8 and 12 weeks respectively. SCA estimates were positive only for CSML×D and CSML×CR at 8 weeks for both AST and ALT, whereas at 12 weeks, CSML×D and D×CR yielded positive SCA estimates. For ALP levels, SCA estimates were positive only for CSML×D and D×CR at 8 weeks, while at 12 weeks all crossbreds resulted positive SCA estimates. Significant SCA variances indicated the presence of non-additive component of variance.
 

 
Hetrosis in serum enzymes
 
At both the ages, heterosis, heterobeltiosis and standard heterosis were positive for AST except for D×CR at 8 weeks for Hbt% (Table 5). For ALT, H% was positive for all the crosses except for CSML×CR at 12 weeks. Except D×CR, Hbt% forall other crosses were positive at 8 weeks, while for 12 weeks, only triple cross resulted in positive Hbt%. Results for Hst% for ALT were similar to those for H%. For ALP, CSML x CR at 8 weeks resulted in negative H%, while all other crosses being positive at both the ages. Hbt% showed similar results at 8 weeks, however, at 12 weeks, triple cross also resulted in negative estimates. Hst% showed similar trend for ALP as noticed earlier for H%. It may be inferred here that, crosses have showed positive values of H%, Hbt% and Hst% in general, which is suggestive of elevated serum enzymes levels in crossbreds than that of their parents, indicating relatively better enzyme profile of purebreds. The literature dealing with serum enzymes levels of crosses in breeding experiment is seldom. Other possible mechanisms involved for these results obtained have already been discussed earlier.
 

Among purebreds, CARI-Red followed by Desi exhibited superior immune responses over CSML, while crossbreds revealed comparable immune response among themselves. Initially, crossbreds exhibited elevated levels of serum enzymes, which reduced in later life, while reverse trend was observed in purebreds. For traits under consideration, both additive and non-additive components of variance were found to play role in their inheritance. Considering this, it may be concluded that, CARI-Red and Desi may be used as improver parent lines for immunocompetence and for hepatic enzymes profile, respectively. On-field cross performance assessment would be a way forward to testify the practicalities for the developed crosses to be used for augmenting rural poultry production.

Authors acknowledge Director, Central Avian Research Institute, Izatnagar and ICAR, New Delhi for providing necessary facilities financial assistance to conduct this study.