Artificial insemination of cryopreserved chicken semen: do different concentration doses affect fertility ?

The relation between fertility and the insemination concentration dose (ID) of cryopreserved chicken semen was studied. Chicken semen was collected from commercial line cockerels housed in single cages and kept according to the standard management guidelines for chicken breeders. Ejaculates were pooled into semen samples and sperm quality was assessed soon after collection. Semen samples were processed for cryopreservation according the following steps: 1) dilution in pre-freezing modified Lake diluent supplemented with trehalose 0.1 M (LD); 2) equilibration at 4°C for 30 min; 3) further dilution in LD added with N-methylacetamide, 6% final concentration, and equilibration for 1 min; 4) loading into 0.25 ml French straws; 5) freezing on a rack floating over a nitrogen bath at 3 cm of height for 10 min; 6) transfer into cryotank for storage. Semen thawing was performed in ice-water bath at 5°C for 100 sec. During semen processing, the correct semen dilution was calculated to reach the following final sperm number per straw corresponding to different IDs: A) 250×106 sperm; B) 500×106 sperm; C) 750×106 sperm. One artificial insemination was performed using semen from one straw per hen. Laying hens (n=27) were divided into 3 groups receiving different IDs according to group A, B and C. Eggs were collected from the second day after AI for 10 days, set every 3/4 days and fertility recorded at candling after 7 days of artificial incubation. All clear eggs were open and true fertility recorded. The overall fertility value recorded in group A, B and C was 6, 8 and 11% respectively and a different fertile period was recorded according to the IDs. In hens receiving 250×106 sperm, fertile eggs were recorded only from day 2 to 4 and the highest fertility value, corresponding to 25%, was recorded on day 3. In contrast, in hens receiving 500×106 and 750×106 sperm, fertile eggs were recorded from day 2 to 10 and the highest fertility values recorded in group B and C were 17 and 20% respectively. In conclusion, the 250×106 sperm ID was suitable to assess in vivo fertility of cryopreserved chicken semen, even if the fertile period was limited to few days after artificial insemination. Increasing the ID to 500 and 750×106 sperm did not improve the fertility rate of cryopreserved chicken semen, but a longer fertile period was recorded.