Clinical Signs and Behavioral Alterations
Clinical signs including alertness of the birds to signal, activity of the birds, crowing behavior which is that the bird is getting mature was noted twice daily. Only the control group showed good signs of maturity while the other birds showed late signs of maturity in a dose gradient manner. The least clinical signs were showed by group D which was fed AFB1 400 ppb (Table 1).
Absolute organ weights
Testes weight was significantly lower in group D as compared to other groups as in Table 2. Groups A, B and C showed non-significant difference with each other. In testes weight of all four groups followed the trend as A> B > C > D. Testes volume was significantly higher in control group A among other groups. In testes volume of all four groups followed the trend as A> B > C > D. Group D has the least testicular volume showing severing testicular atrophy by the AFB1 400ppb fed to the birds in that group.
In 2nd killing testes weight was significantly higher in control group A. Group D testes weight was significantly lower as compared to control group. Testes volume group A was significantly higher compared to group D while non-significant with group B and C.
Relative organ weight of WLH male layers
In the 1st killing the relative organs weight of testes of group D was significantly lower as compared to control group A. The trend seen in testes weight were as follows (A > B > C > D). In second killing there was no significant change in the relative weight of the testes in all four groups however control group has maximum relative testes weight and group D has least relative testes weight. There was no significant change in the relative volume of the testes in all four groups however control group has maximum relative testes volume and group D has least relative testes volume Table 3.
Antibody titers against SRBC
Titers at 7th Day of Primary Dose
Antibody titer of total antibodies at the 7th day of the initial dose of group A and group B was non-significant with each other and significantly higher as compared to groups C and D while the group D showed the lowest antibody titer on the 7th day. The IgM titers were non-significant among all groups but group D showed the least value of IgM and group A showed the maximum value of IgM on 7th day. The IgG titers of group B were significantly higher as compared to groups C and D while group D showed the least antibody titer for IgG (Figure a1).
Total Antibody titers on the 14th day of group A were significantly higher as compared to group C while groups C and D were significantly lower as compared to the control group. The IgM titer of group A was significantly higher as compared to group C. The IgG titers were significantly higher in group A as compared to groups C and D. groups A and B were non-significant with each other while group D showed the least value. Antibody titers at the 7th day of a booster dose of group A were significantly higher as compared to groups B, C and D. While the group D showed the minimum value. The IgM titer of group A was significantly higher as compared to other groups. The IgG titers of group D were significantly lower as compared to control group A.
On the 14th day of a booster dose, the total antibody titers of group A were significantly higher than all other groups with group D showing the minimum levels of total antibodies. The IgM titers were non-significant in all groups but group A showed the maximum titer for IgM as compared to other groups. The IgG titer of group A was significantly higher as compared to other groups as shown in figure b1.
Mononuclear Phagocytic Response Carbon Clearance Assay
In-vivo phagocytic response of mononuclear cells to carbon particles at 3 minutes response groups had non-significant difference among all groups. At 15 minutes, group D was having significantly higher absorbance as compared to the control group and all other groups. The control group was significantly lower compared to other groups. All the groups followed a trend such as group (D > C > B > A) which means the phagocytic activity in the group D was least to clear the carbon injected into the bloodstream showing a weaker immune response while the lowest absorbance value at response 15 minute shown by control group means that the immune system was functioning very well and responded significantly by clearing the carbon injected into the body by the phagocytic activity shown by the mononuclear cells (Fig. 2).
Lymphoproliferative response to avian tuberculin
At 24 hours, control group (A) showed a response that was significantly higher as compared to all other groups especially group D. While group D was significantly lower as compared to group B and group B and group C were significantly different from each other at 24 hours response. At response time 48 hours control group A was significantly higher as compared to all groups. Group B and group C were non-significant to each other while both groups showed response significantly higher as compared to group D. At response 72 hours group A, B and C did not show significant difference with each other, however, the lymphoproliferative response of group D was significantly lesser compared to the control group (Fig. 3).
Sperm Motility Percentage
Sperm motility was decreased in all groups with increasing doses of toxin as shown in Table 4.
Serum levels of these reproductive hormones (testosterone) clearly follow the trends which are Group A> B >C >D. This means that the level of this hormone decreased as there was an increase in the level of AFB1 in their diet as shown in Table 5. The level of luteinizing hormone did not show any significant increase in its level but the level of prolactin was increased significantly with increase in dose of toxin.
There was no obvious gross lesion seen in the control group. Testes showed normal size and weight. However, there was a significant decrease in testicular size in a dose-dependent manner between the three groups fed with AFB1 contaminated feed (Fig. 4).
Seminiferous tubules in control group A showed normal appearance. The lumen of the tubule was patent. Spermatogenesis developmental stages such as spermatogonia, spermatocytes, spermatids and mature spermatozoa were found. The presence of clusters of spermatozoa indicated normal testicular parenchyma (Fig.5a &b). Testicular parenchyma in group B also showed normal appearing seminiferous tubules. All the morphological stages of spermatogenesis i.e. spermatogonia, spermatids, spermatocytes and spermatozoa were visible. However, in some of the seminiferous tubules partial arrest of spermatogenesis was present (Fig. 5c&d). Likewise, all the stages of spermatogenesis were present in group C. But the number of spermatozoa was less indicating partial arrest of spermatogenesis. In a few places, the lumen of the seminiferous tubule was not patent (Fig. 5c&d). In group D, arrested spermatogenesis was evident in many areas as the lumen of the seminiferous tubules was not patent. Developmental stages of spermatogenesis were seen except the mature spermatozoa. In some tubules, few spermatozoa were also present. Number of spermatozoa was less; necrotic changes were also present in seminiferous tubule (Fig. 5e & f).