Pakistan has vast population of dogs belonging to different breeds. Most of the dogs have no pedigree record which is a great threat to conservation of different breeds. No study on DNA fingerprinting of dogs has been conducted in Pakistan. DNA fingerprinting of dogs is necessary to overcome the problems like forensic cases, sale & purchase, individual identity in case of fertilization by more than one male and ownership disputes. Microsatellite markers have been proved as an efficient and powerful tool for parentage testing and breed characterization of dogs. In this study, a panel of microsatellite markers, having high polymorphism information content (PlC) values, was developed. Blood samples were taken from cephalic vein of two breeds of dogs (German shepherd and Labrador retriever). DNA was extracted by Inorganic method. Primers of microsatellite markers were optimized for successful amplification conditions in the Bio-Rad thermocycler. Multiplex PCR was performed, for amplification of these microsatellite markers on 46 samples belonging to 20 families. Genotyping analysis was performed for the PCR products of microsatellite markers on non denaturing polyacrylamide gel. These results were analyzed statistically software "POPGENE 3.3 and POWER STAT". Allele frequency, heterozygosity, homozygosity, polymorphism information content (PlC), power of discrimination and power of exclusion of all microsatellite markers were calculated. Average power of discrimination among non parents, average hetrozygosity, average observed homozygosity and average polymorphism information content (PlC) value for all alleles was 0.809, 0.6345, 0.29 13 and 0.724 respectively. Moreover combined power of exclusion reached a significant value of 0.9998. Almost all of the microsatellite markers showed significant variations in both German shepherd and Labrador retriever breeds. Microsatellite "REN41D2Ob" showed maximum variation i.e. 17 alleles and microsatellite"REN49F22b" showed the least variation among all microsatellite markers i.e. 4 alleles. Genotyping results of microsatellite markers were clearly different for two different breeds showing a distinct genetic distance between German shepherd and Labrador retriever breeds.
Results of this study lead to development of a panel of microsatellite markers which can be used for parentage analysis and breed characterization of dogs. This was a preliminary study on dogs in Pakistan. This facility can be provided on commercial basis to pet owners and kennel clubs. Moreover this study can become the basis for further research investigations in canines in Pakistan.

To evaluate effects of various stress factors on immune response and growth performance of broiler chicks, a total of five experiments using 2000 broiler chicks were conducted. In each experiment, chicks were divided into five groups (A, B, C, D and E), and each group consisted of 80 day-old-chicks. In each experiment, the chicks were exposed to stress factors, such as temperature, stocking density, feed deprivation, water restriction and light. Each chick in groups A, B, C, and E was vaccinated against IBV, NDV, IBDV and HPSV, but chicks in group D were kept as unvaccinated controls. Blood samples from each group were collected on 36 day of age, at 18 hrs for determining TLC, DLC and H/L ratio. The antibody titers of chicks in different groups were analyzed using HI test at days 36th and 56. The cellular response was analyzed by injecting PHA-P in the wattles of bird during post stress period. The effects of each stress on lymphoid organs were determined. The potential to resist virulent NDV challenge and effect of stress factors on body weight gains and FCR of chicks was also determined.
In experiment 1, conducted to determine the effect of various temperature ranges on broiler chicks, it was observed that the heat stressed (HS) birds showed non-signilicant dilYerence in TLC values. The HS effect on lymphoid organs indicated that the mean weight of thymus of chicks in group B (0.29±0.02) and C (0.59±0.13) was significantly (P<0.05). lower than those in groups A (4.11±3.26), D (4.50±0.77) and E (4.35±0.21). The mean bursa weight of heat stressed chicks in goups A (0.94±0.59) and B (0.20±0.01) were significantly (P0.05) lower as compared to non- heat stressed chicks in groups D (1.42±0.22) and E (1.33±0.18). The mean spleen weight of groups A (1.21±0.13) and B (1.30±0.11) was significantly (P0.05) lower than groups D (L93±0.16) and E (1.52±0.10) indicating the adverse effect of increased temperature. The FCR valueswere significantly (P0.05) different among groups in 6th1 week and effect of Vitamin C was found significantly (P<0.05) improved than Vitamin E and glucose treatment. At 36 day of age the HI titer was recorded significantly (P<0.05) lower in group A (GMT 61) than B (GMT 144) and C (GMT 109) groups while group E (GMT 186) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 days was 79, 156, 122 and 216, respectively. There was nonsignificant (P>0.05) differences in wattle thickness (cm) among groups A (1.51+0.06), B (1.77±0.26), C (1.2±0.25) and D (1.52±0.22) but increased in group E (1.83+0.08). The mortality was found significantly (P<0.05) higher in groups A (14) and D (40) on challenge with NDV virulent virus.
In experiment 2, conducted to determine the effect of various levels of stocking densities on broiler chicks, it was observed that stressed birds had showed non-signilicant (P>0.05) difference in TLC values. The effect on lymphoid organs found that the mean thymLls weight (grn) of chicks in groups A (0.37±0.04) and B (0.74±0.17) were significantly (P<0.05) lower than groups C (1.50±0.35), D (4.43±0.72) and E (4.40±0.23). The mean bursa weight (gm) of groups A (0.33+0.03) and B (0.57+0. 1 7) were significantly (P0.05) lower than those of groups D (1.76±0.05) and 13(1.33±0.08) indicating that less space effect the bursa development in the chicks. The mean spleen weight (gm) of groups A (1.18±0.07) and B (1.52±0.20) was significantly (P<0.05) lower than group D (2.37±0.28). The FCR values were sign ilicantly (P<0.05) different among groups in 6thi week and there was non-signiflcant (P>0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose. At 36th day of age the I-Il titer was recorded significantly (P<0.05) lower in group A (GMT 67) than groups B (GMT 9.6) and C (GMT 102). The chicks in group D (GMT 07) showed negligible HI titer while group E (GMT 185) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the 1-Il antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease indicating that the titers in the birds were not enough to resist the virulent challenge. The postNDV-challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 86, 121, 132 and 210. There wa non-significant (P0.05) differences in wattle thickness (cm) in groups A (1.44±0.07) and C (1.43±0.10) while group E (1.64±0.31) showed significantly (P0.05) increased wattle thickness. The mortality was found significantly (P0.05) higher in groups A (25) and D (40) on challenge with NDV virulent virus. This indicated that less floor space decreased the immune response of the birds which leads to the infection/death.
In experiment 3, effect of feed deprivation at different time intervals on broiler chicks was studied, it was observed that feed deprivation stressed birds had showed non-significant (P>0.05) differences in blood cells population. The effect of feed deprivation on the mean thymus weight (gm) of chicks in group C (0.96±0.29) was adversely affected as the chicks in this group had significantly (P<0.05) lower weight than groups B, 1) and E. The bursa mean weight (gm) of groups A (0.48±0.11) and C (0.52±0.06) was significantly (P0.05) lower than those of groups D (1.40±0.18) and E (1.28±0.11) indicating, that 24 hrs and morning off-feed effect the bursa development in the chicks. The mean splen weight(gm) of groups A (I. 19±0.07) and C (1.21±0.06) vcre significantly (P0.05) lower than groups D and E indicating adverse effect ol24hrs and day off feed on chicks lead to infection. The FCR values were significantly (P0.05) different among groups in 6hhl week and there was significant (P<0.05) differences among groups with treatment of glucose than Vitamin C and Vitamin E treated. groups. At 36th day of age, the HI titer was recorded significantly (P<0.05) lower in group B (GMT 15) than C (GMT 74) and group D (GMT 07) showed negligible HI titer while group E (GMT 140) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 68, 54, 115 and 165. There was non-significant (P0.05) difference in wattle thickness (cm) among groups while group E (I .78±0.06) showed significantly (P<0.05) increased wattle thickness. The mortality was Found significantly (P<0.05) higher in Groups C (12) and D (40) on challenge with NDV virulent virus. This indicated that 24 hrs off feed decreased the immune response of the birds which leads to the infection.
In experiment 4, studied the effect of water restriction at different time intervals on broiler chicks, it was observed that water restricted birds had nonsignificant (P0.05) differences in blood cells population except lymphocytes percentage was found higher in groups A (43.7±2.47), C (43.7±1.16) and D (53 .3±1 .30) than group B (39.1±1.06). The effect of water restriction on the mean thymus weight (gm) of chicks in group C (0.60±0.07) was adversely effected as the chicks in this group had significantly (PO.O5) lower weight than group D (4.09±0.70) indicating that increased in the period of water restriction in chicks adversely affected the mean thymus weight and chicks reared on ad-flbituni water had higher mean thymus weight. The mean bursa weight (gui) of group C (0.07±0.02) was significantly (P<0.05) lower as compared to group D (1.37±0.88) indicating that water restriction of 24 hr had affected the bursa development in the chicks. The mean spleen weight (gm) of group C (2.64±1.49) was significantly (P0.05) higher than groups A, B and E. The FCR values were significantly (P<0.05) different among groups in 6th week and there was non-significant (P0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose treated groups. At 36th day of age the HI titer was recorded significantly (P<0.05) lower in group D (GMT 09) than A (GMT 54) and group E (GMT 109) showed significantly (P0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 clay was 78, 63, 48 and 134. There was non-significant (P0.05) difference in wattle thickness (cm) among groups B and E and group A (1.28±0.08) showed significantly (P0.05) lower wattle thickness. The mortality was found significantly (P<0.05) higher in groups B (14) and C (21) on challenge with NDV virulent virus. This indicated that 18 and 24 hrs water restriction decreased the immune response of the birds.
In experiment 5, effect of light stress at various time intervals on broiler chicks was studied. It was observed that light stressed birds had showed non-significant (P>0.05) difference on blood cells population except lymphocytes percentage was found higher in groups D (6 1.4±1.16) than group C. The effect on lymphoid organs studied and the mean thymus weight (gin) old chicks in group B (I .90±0.53) was adversely effected as the chicks in this group had significantly (P0.05) lower mean thymus weight than groups D (4.64±0.74) and E (4.34±0.25) indicating that increase in the period of oil-light in chicks adversely effected the mean thymus weight and chicks reared on 24hr light had higher mean thymus weight. The bursa mean weight (gm) 01' group 13(0.43±0.05) was significantly (P0.05) lower as compared to group D (1.59±0.17). The spleen mean weight (gun) of group D (1.88±0.15) was significantly (P0.05) difference in wattle thickness (cm) among groups while group B (1.53±0.15) showed significantly (P<0.05) increased wattle thickness. The mortality was found significantly (PO.05) higher in groups A (18) and D (40) on challenge with NDV virulent virus. This indicated that 24 hr off-light decreased the immune response of the birds.