Cryoprotectant effects of egg yolk on Indian red jungle fowl (Gallus gallus murghi) sperm.

Egg yolk is a good external cryoprotectant of mammalian sperm and some wild bird's sperm, but, at least in domestic breeds of chicken (Gallus gallus domesticus), it may inhibit eventual fertilization of ova when high concentrations are used. We hypothesized that egg yolk can protect the sperm from cryo-induced damages providing adequate fertilization in one phylogenetic wild ancestor of current chicken breeds: the Indian red jungle fowl (Gallus gallus murghi). To test the hypothesis, the present study was designed to evaluate different concentrations of egg yolk in extender in comparison with glycerol. Semen collected from Indian red jungle fowl cocks (n = 8) was cryopreserved using different egg yolk concentrations (10%, 15%, 20% and 25%) or 20% glycerol (control group) following routine protocol of cryopreservation. During cryopreservation, sperm motility (67.5 ± 2.5%), plasma membrane integrity (66.3 ± 2.4%), viability (58.8 ± 1.3%) and acrosomal integrity (60.0.8 ± 2.0%) were recorded highest in an extender with 15% egg yolk compared to other experimental extenders and control at post-dilution, cooling, equilibration and thawing. The no. of fertilized eggs (26.6 ± 0.7, 21.6 ± 1.2), percent fertility (55.9 ± 4.4, 46.5 ± 2.2), no. of hatched chicks (23.6 ± 1.0, 17.2 ± 1.0), percent hatch (49.5 ± 3.2, 37.1 ± 2.5%) and hatchability of the fertile eggs (89.4 ± 2.2, 79.7 ± 3.7) were recorded higher (P < 0.05) with semen cryopreserved with 15% egg yolk compared to control (20% glycerol). It is concluded that 15% egg yolk can be used in cryopreservation protocol of Indian red jungle fowl sperm.

Rakha BA ，Ansari MS ，Akhter S ，Santiago-Moreno J ，Blesbois E ... -  《-》

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen.

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°C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37°C to 4°C @ -0.275°C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10min, filled in 0.5mL French straws, kept over LN2 vapours for 10min and plunged into LN2 and stored at -196°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°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.

Rakha BA ，Ansari MS ，Akhter S ，Hussain I ，Blesbois E ... -  《-》

Dimethyleacetamide improves the cryosurvivability of Indian red jungle fowl (Gallus gallus murghi) sperm.

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol.

Rakha BA ，Ansari MS ，Akhter S ，Zafar Z ，Naseer A ，Hussain I ，Santiago-Moreno J ，Blesbois E ... -  《-》

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone.

The Indian red jungle fowl is a sub-species of the genus Gallus native to South Asia; facing high risk of extinction in its native habitat. During cryopreservation, permeable cryoprotectants like glycerol are usually employed and we previously showed encouraging results with 20% glycerol. Because bird spermatozoa contain very little intracellular water, the possibility of replacing an internal cryoprotectant by an external one is opened. In the present study, we tested the replacement of internal cryoprotectant glycerol by the external cryoprotectant Polyvinylpyrrolidone (PVP). PVP is a non-permeable cryoprotectant and keeps the sperm in glassy state both in cooling and warming stages without making ice crystallization within the sperm cell. We evaluated the effect of various levels of polyvinylpyrrolidone (PVP) on Indian red jungle fowl semen quality and fertility outcomes. The qualifying semen ejaculates collected from eight mature cocks were pooled, divided into five aliquots, diluted (37 °C) with red fowl semen extender having PVP [0% (control) 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v)]. Diluted semen was cryopreserved and stored in liquid nitrogen. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) with 6% PVP at post-dilution, cooling, equilibration and freeze-thawing. Higher (P < 0.05) no. of fertile eggs, fertility, no. of hatched chicks, percent hatch and hatchability was recorded with 6% PVP compared to control. It is concluded that 6% PVP maintained better post-taw quality and fertility of Indian red jungle fowl spermatozoa than glycerol and can be used in routine practice avoiding the contraceptive effects of glycerol.

Rakha BA ，Ansari MS ，Akhter S ，Zafar Z ，Hussain I ，Santiago-Moreno J ，Blesbois E ... -  《-》

Use of dimethylsulfoxide for semen cryopreservation in Indian red jungle fowl (Gallus gallus murghi).

Glycerol is a least toxic and most effective cryoprotectant for cryopreservation of poultry semen, but due to its contraceptive properties removal of glycerol is usually needed prior to artificial insemination. Dimethylsulfoxide (DMSO), a small amphiphilic molecule used as penetrating cryoprotectant for biological cells, has been recognized as an adequate alternative for cryopreservation of sperm from several species. This study was designed to evaluate the efficacy of different concentrations of DMSO as cryoprotectant for Indian red jungle fowl (Gallus gallus murghi) sperm. Semen was collected from Indian red jungle fowl cocks, pooled and divided into five aliquots. Different concentrations of DMSO (0%, 4%, 6%, 8% and 10%) were compared. Diluted semen was cooled from 37 °C to 4 °C (-0.275 °C min-1), 20% glycerol added to control and equilibrated for 10 min. After equilibration, semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Semen samples were thawed at 37 °C for 30 s. Cryo-survival of Indian red jungle fowl sperm was affected by cryopreservation stages and different concentrations of cryoprotectant used. Highest sperm motility (85.0 ± 2.9; 80.0 ± 3.5; 71.3 ± 4.3; 60.0 ± 1.3), plasma membrane integrity (79.5 ± 3.8; 75.3 ± 2.4; 72.8 ± 3.3; 60.3 ± 2.8), viability (80.8 ± 4.6; 75.5 ± 2.9; 71.0 ± 7.6; 58.8 ± 1.3) and acrosomal integrity (76.3 ± 2.4; 72.0 ± 6.0; 62.5 ± 4.3; 55.0 ± 3.2) were recorded in a diluent having 8% DMSO at post-dilution, cooling, equilibration and freeze-thawing. Highest fertility results were obtained after artificial insemination with 8% DMSO compared to 20% glycerol (73.0 ± 4.4 vs 53.1 ± 4.3, P < 0.05). It is concluded that 8% DMSO as a permeable cryoprotectant improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

Rakha BA ，Ansari MS ，Akhter S ，Zafar Z ，Naseer A ，Hussain I ，Blesbois E ，Santiago-Moreno J ... -  《-》