The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl (Gallus gallus murghi) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37 degrees C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37 degrees C to 4 degrees C@ -0.275 degrees C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10 min, filled in 0.5 mL French straws, kept over LN2 vapours for 10 min and plunged into LN2 and stored at 196 degrees C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (P <0.05) in red fowl extender at 0, 2 and 4h of incubation post-thaw. After cryopreservation and post-thawing at 37 degrees C the highest (P <0.05) recovery rates and absolute livability index was also recorded in red fowl extender that was thus used for further artificial insemination of cooled-diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs (Liquid, 20.6 +/- 0.4; Cryopreserved, 12.6 +/- 0.5), percent fertility (86.7 +/- 2.2; 57.2 +/- 3.9), no. of hatched chicks (18.2 +/- 0.8; 10.0 +/- 0.3), percent hatch (76.5 +/- 2.7; 45.3 +/- 2.2) and hatchability of fertilized eggs (88.3 +/- 3.4; 79.6 +/- 3.4) were higher with sperm respectively freshly cooled-diluted or cryopreserved in red fowl extender. However, the rates obtained with frozen-thawed sperm were already successful for cryo-banking purpose and artificial insemination practice. In conclusion, we show the first fertility success obtained with cryopreserved Indian jungle fowl sperm. In addition, the red fowl extender is superior in maintaining the quality of Indian red jungle fowl cryopreserved sperm compared to Beltsville poultry, Lake, EK, Tselutin poultry and chicken semen extender. (C) 2016 Elsevier B.V. All rights reserved.
