Fowl Spermatozoa Research Articles

Effect of ascorbic acid on metabolic status, lipid peroxidation, antioxidant activity and quality of frozen Indian red jungle fowl (Gallus gallus murghi) semen.

The Indian red jungle fowl population is decreasing in its natural habitat. Its conservation through semen cryopreservation with sufficient live sperm recovery rate is requisite where ascorbic acid could play significant role to mitigate cryo-incited injuries. The objective was to elucidate the effect of ascorbic acid on freezability of Indian red jungle fowl sperm. Pooled semen was aliquoted and diluted (1:5) with red fowl extender having ascorbic acid: 0.0 (control), 1.0, 2.0 and 4.0 mM. Diluted samples were cryopreserved and semen quality was assessed at post-dilution, cooling, equilibration and freeze-thawing stages. Sperm metabolic status, antioxidant potential and lipid peroxidation were studied at post-dilution and freeze-thawing. Sperm motility did not differ (p > .05) in experimental extenders and control at post-dilution and cooling; however, it was recorded higher (p < .05) with ascorbic acid at 2.0 mM compared with other levels at post-equilibration and post-thawing stage. Sperm viability, plasma membrane and acrosome intactness were recorded higher (p < .05) with 2.0 mM ascorbic acid compared with other concentrations of ascorbic acid at all stages of cryopreservation. Sperm metabolic status and antioxidant potential were recorded higher (p < .05), while lipid peroxidation was recorded lowest (p < .05) with 2.0 mM ascorbic acid compared with 1.0, 4.0 mM and control. In conclusion, ascorbic acid at 2.0 mM in red fowl extender improves quality, metabolic status and antioxidant potential of frozen Indian red jungle fowl semen through ameliorating lipid peroxidation.

The basis of cell cryopreservation

Cryopreservation (banking) techniques have been known to nature for centuries. Many species of insects, amphibians, fish and even reptiles use natural cryopreservation methods to survive the harsh conditions of winter or to live in extremely cold temperatures. Cryopreservation and dreams of immortality have intrigued humanity for years. The first reports of observing the effects of freezing sperm (stored in snow) date back to 1776. In 1866, Montegazza was the first to suggest a vision completely unimaginable for the time: "a man dying on the battlefield can conceive an heir from sperm frozen and stored at home". The first, at that time still unsuccessful, reports of laboratory freezing of human sperm date back to the 1930s [1]. Finally, mankind "learned" cryopreservation in the middle of the twentieth century, when on October 15, 1949, the article "Revival of spermatozoa after vitrification and dehydration at low temperatures" appeared in print in the Nature journal, summarising the pioneering research of scientists from the National Institute for Medical Research, Mill Hill, London [2]. This concerned the freezing of fowl sperm in the presence of glycerol, ethylene glycol and propylene glycol in such a way that after thawing it was able to fertilise eggs effectively. The subsequent use of dimethyl sulfoxide (DMSO) revolutionised modern cryobiology [3-5]. Thus began the era of cryopreservation, without which today it is difficult to imagine the work of cell biology laboratories, modern animal breeding, or the development of modern medicine.

Effect of glutathione on pre and post-freezing sperm quality of Indian red jungle fowl (Gallus gallus murghi)

During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules and insufficient natural antioxidant defence system. Therefore, present study was designed to identify the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi) sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen was divided in four aliquots, diluted with red fowl extender (1:5) at 37 °C having GSH 0 mM (control), 0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (−196 °C) in liquid nitrogen. Semen quality was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at 37 °C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling, equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scavenging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integrity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle fowl through enriching antioxidant potential and ameliorating the oxidative stress.

Effect of Quercetin on Oxidative Stress, Mitochondrial Activity, and Quality of Indian Red Jungle Fowl (Gallus gallus murghi) Sperm.

Aim: The study was designed to elucidate the effects of quercetin in an extender on oxidative stress, mitochondrial activity and quality of Indian red jungle fowl (Gallus gallus murghi) sperm during cryopreservation. Materials and Methods: Semen was collected from seven adult males through abdominal massage and evaluated for semen volume, concentration, and motility. The qualifying semen ejaculates having >80% motility were diluted in red fowl extenders with 0 (control), 5, 10, 15, and 20 mM quercetin. Diluted semen was frozen following a glycerol-based protocol. Semen quality (motility, plasma membrane integrity, viability, acrosome integrity, and chromatin condensation status) and biochemical parameters (mitochondrial activity, ferric reducing antioxidant power, and malondialdehyde [MDA]) were determined at various stages of cryopreservation. Results: Sperm motility, plasma membrane integrity, viability, acrosome integrity, and chromatin condensation were recorded highest (p < 0.05) with 15 mM quercetin compared with 5, 10, and 20 mM quercetin and control at post-dilution, cooling, equilibration, and freeze-thawing. Nevertheless, mitochondrial activity and antioxidant potential were recorded highest with 15 mM quercetin compared with all experimental extenders at post-equilibration and freeze-thawing. MDA concentration in sperm and seminal plasma were recorded lowest (p < 0.05) in the extender having 15 mM quercetin at post-equilibration and freeze-thawing. Cryopreservation stages showed negative effects (p < 0.05) on semen quality parameters, irrespective of experimental extenders. Conclusions: It is concluded that quercetin (15 mM) supplementation in red fowl extender improves sperm motility, plasma membrane integrity, viability, acrosome integrity, chromatin condensation, and mitochondrial activity by elevating the total antioxidant potential and ameliorating lipid peroxidation during cryopreservation.

Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi)

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender 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.

PSXIII-4 Species feature of morphometric parameters in poultry semen

Abstract The aim of the research was to study the specific features of morphometric parameters in rooster and guinea fowl spermatozoa. Thirty ejaculate samples from each species were investigated. A macroscopic assessment of the ejaculates obtained was carried out by volume, color and consistency. Microscopic parameters were studied using computerized sperm quality analysis systems. Diff-Quick set was used for staining sperm. Visualization of fixed preparations was performed using a Nikon Eclipse Ni-U microscope and a Nikon DS-Qi2 camera. Measurement of morphometric parameters was carried out in the program NIS-Elements BR 4.30. Evaluation of sperm morphometry was carried out according to the following indicators: total length of spermatozoa, length of head and tail, perimeter and area, head shape in indexes of ellipticity and elongation. The SPSS v.15.0 package was used for statistical analysis. To assess the statistical differences in the distributions of spermatozoa, the χ2-test was used. ANOVA was conducted to determine the effect of species. Roosters surpassed guinea fowls in ejaculate volume by 83.3% (P &amp;lt; 0.001). Statistically significant differences were not established for sperm activity between species. The content of motile spermatozoa in the roosters ejaculate was 92.4 ± 5.3%, in guinea fowls - 88.7 ± 6.54%. The results of the morphometric parameters analysis showed that they have a specific feature. The total length of rooster spermatozoa was 90 ± 2.6 μm, which is 8 μm more than that in guinea fowl (P &amp;lt; 0.05). The length of the sperm head in roosters was 2 μm longer than that in guinea fowls (P &amp;lt; 0.05). The interspecific difference across the perimeters and width of the head was not statistically significant. Rooster ejaculates differed relative homogeneity compared to guinea fowl. The study results showed that morphometric parameters of poultry sperm have a specific feature. Supported by the RSF (16-16-04104).

Main signaling pathways involved in the control of fowl sperm motility.

Motility is an essential function of the mature male gamete and is widely used as an indicator of semen quality. Knowledge of signal transduction pathways regulating sperm motility is therefore important to gain insight into new methods to increase the fertilizing ability of semen samples, especially those subjected to biotechnological processes such as cryopreservation. In this article, we will briefly describe the roles of several signaling pathways involved in the motility of fowl sperm as compared to mammalian sperm. This review is intended to facilitate the design of future studies in fowl male fertility and improve the quality of frozen-thawed sperm.

Effect of Cryopreservation on Lipid Peroxidation, Antioxidant Potential, Chromatin Integrity, and Mitochondrial Activity of Indian Red Jungle Fowl (Gallus gallus murghi) Semen.

Aim: The study elucidates the impact of cryopreservation on lipid peroxidation (LPO), antioxidant potential, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm. Materials and Methods: Semen from eight mature cocks was pooled and diluted at 37°C using a specific medium for red fowl species. The diluted semen samples were immediately refrigerated at 4°C for 2 hours (0.275°C min-1). Glycerol was then added to a final concentration of 20%, and the samples were equilibrated for 10 minutes at 5°C before loading into 0.25 mL French straws. These straws were then frozen by placing them in liquid nitrogen vapor for 10 minutes and plunged into liquid nitrogen. Motility, viability, DNA integrity, antioxidant activity, and LPO were assessed before dilution (fresh semen), after equilibration (processed semen), and post-thawing (frozen semen). Results: Sperm motility, viability, DNA integrity, and mitochondrial activity decreased (p < 0.05) in processed and frozen semen compared with fresh semen. Nevertheless, the concentration of malondialdehyde (MDA) in sperm and seminal plasma was greater (p < 0.05) in frozen-thawed and processed semen compared with fresh semen. Multivariate regression analysis showed a negative impact of MDA concentration in sperm (R2 = 0.90, Wilk's λ = 0.003, p < 0.001) and seminal plasma (R2 = 0.84, Wilk's λ = 0.02, p < 0.001) on motility, viability, DNA integrity, and mitochondrial activity. Nonetheless, total antioxidant capacity (TAC) had a positive impact on sperm variables (R2 = 0.82, Wilk's λ = 0.096, p < 0.001). Conclusions: The decrease in motility, viability, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm was associated with an increase in LPO during cryopreservation. Furthermore, TAC was reduced during the freeze-thaw process, which was insufficient in protecting the sperm against high reactive oxygen species levels.

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.

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.

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.

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.

Cryopreservation of Indian Red Jungle Fowl (Gallus Gallus Murghi) Spermatozoa: A First Approach

The experiment was designed to show the capacity of Indian Red Jungle Fowl ( Gallus gallus murghi) sperm to be cryopreserved. The changes in characteristics of Indian Red Jungle Fowl spermatozoa during different phases of cryopreservation with the glycerol cryoprotectant and straw packaging were recorded. Semen from eight mature cocks were pooled, diluted at 37 °C in a Tselutin poultry extender, cooled to 4 °C in 2 h (0.275 min−1) and equilibrated for 10 min after the addition of 11% glycerol. Cooled semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapour for 10 min and plunged into liquid nitrogen. Frozen semen was thawed at 37 °C for 30 s. Sperm motility, plasma membrane plasticity, livability, and acrosomal integrity were assessed at the stage of pre-dilution (37 °C), post-dilution, cooling, equilibration and freeze-thawing. The experiment was repeated five times. Sperm motility did not change at post-dilution and cooling and averaged 83.3±3.1%. However, motility declined ( P&lt;0.05) to 66.0±2.4% and 45.0±6.5% at post-equilibration and thawing stages, respectively. The sperm plasma membrane plasticity at the pre-dilution stage was 74.8±1.4% and decreased ( P&lt;0.05) to 62.3±1.7%, 51.7±1.7%, and 42.0±1.2% at the stages of post-dilution, cooling and freeze-thawing, respectively. A decline ( P &lt; 0.05) in acrosomal integrity was observed at post-cooling (83.8±2.4%), equilibration (80.8±0.8%) and thawing (73.8±2.4%). It is concluded that Indian Red Jungle sperm seem to adapt quite well to the freeze-thaw process that mainly alters the motility capacities and much less the acrosomal integrity. To our knowledge, this is the first ever report on cryopreservation of Indian Red Jungle Fowl spermatozoa.

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.

Comparison of Extenders for Liquid Storage of Indian Red Jungle Fowl (Gallus Gallus Murghi) Spermatozoa

This study was designed to evaluate a range of avian semen extenders for liquid storage of Indian Red Jungle Fowl ( Gallus gallus murghi) spermatozoa at 5 °C. Semen was collected from 8 mature trained cocks and processed in the Beltsville Poultry, Turkey, Lake, EK, Tselutin Poultry and Chicken semen extenders for storage at 5 °C. Semen quality parameters viz, motility (%), plasma membrane integrity (%), livability (%) and acrosomal integrity (%) were assessed at 0, 3, 6, 24 and 48 hours of storage. A time dependent decrease was observed in motility, plasma membrane and acrosomal integrity in all experimental extenders during the storage period. However, the Turkey semen extender was found significantly ( P&lt;0.05) superior for protecting all aforementioned semen quality parameters compared to the Beltsville Poultry, Lake, EK, Tselutin Poultry and Chicken semen extenders. It is concluded that the Turkey semen extender can be used efficiently for the liquid storage of Indian Red Jungle Fowl spermatozoa at 5 °C.

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-β-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl spermatozoa.

Visible Diode Laser Enhancement of Exotic DNA Uptake by Fowl Sperm

An experiment was conducted to assess the use of low power laser irradiation and lipofectin to enhance fowl sperm uptake of exogenous DNA. Semen samples of 10 roosters were collected and pooled. The pooled sample was diluted with a semen extender and then divided into 6 aliquots for 6 different treatments. The treatments included different combinations of the inclusion of exogenous DNA (bacterial plasmid pUC18), the exposure to low power laser irradiation and the transfection with lipofectin (5%). Laser irradiation was by using visible diode laser (650 nm) at energy dose of 4 J/cm2. The recognition of the plasmid DNA in the sperm was by using two specific oligonucleotides (forward and reverse) to prime a 420-bp fragment on the pMB1 rep of the plasmid. The results indicated that low power laser irradiation enhanced the sperm uptake of the plasmid DNA. Also, lipofectin enhanced the introduction of the plasmid DNA into the sperm, whether the semen was laser irradiated or not.

Premises for fowl sperm preservation based on applied bioenergetics1

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

An efficient method of guinea fowl sperm cryopreservation

France is the only country that practices pedigree selection of guinea fowl for meat production. The increasing risk of line extinction for sanitary or breeding failure reasons makes clear the need for an efficient method of reproductive cell cryopreservation in this species. However, an efficient method of guinea fowl sperm freezing in secured packaging is still lacking. The aim of the present study was to develop such a method. Based on results previously obtained in chickens, different cryoprotectants and freezing/thawing processes were tested and then adapted to guinea fowl. Semen quality was measured by semen viability evaluation and then by fertility measured after intravaginal artificial insemination. The best results (70% fertility with frozen-thawed sperm) were obtained by the use of the permeant cryoprotectant agents dimethyl formamide combined with a freezing rate of 30°C/min. The initial insemination frequency also affected the fertility results: 2 consecutive days of inseminations were needed in the first week to ensure enough filling of the utero-vaginal glands of the guinea fowl hen and thus to get successive fertile eggs. Thereafter, a 2-wk insemination frequency was sufficient. This new method, combining biophysical (cryoprotectant agents, freeze/thaw rate) and zootechnical (artificial insemination frequency) features, is the first cryopreservation method successfully developed in secured packaging for guinea fowl sperm. This method is now available for the practice of gene bank conservation and male reproductive management.

Short-term preservation of fowl sperm in buffered potassium chloride

Previous research demonstrated that sperm motility is dependent upon mitochondrial calcium cycling. Thus, sperm are inactivated when extracellular calcium ions are chelated. Mitochondrial calcium cycling, however, is driven by extracellular sodium ions. The hypothesis that sperm inactivation is subject to 2 variables was tested in the present work. Sperm motility was evaluated with computer-assisted sperm motion analysis in the first experiment. Sperm became immotile within minutes when suspended in buffered isotonic potassium chloride containing calcium ions. This outcome set the stage for the second experiment in which sperm were inactivated by centrifugation through 12% (wt/vol) Accudenz prepared with potassium chloride and tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Sperm mobility was the end point in the second and subsequent experiments. Potassium chloride was a suitable medium in regard to sperm inactivation with BAPTA followed by storage for 2 h at 10°C. Consequently, sperm so inactivated were reactivated after 1, 2, 3, 4, and 5 h of storage in the third experiment. Whereas pre- and postwash sperm mobility were equivalent, sperm mobility declined with time (P < 0.001) without exogenous energy in the storage medium. Therefore, the effect of 5 mM glucose was tested in the fourth experiment. In this case, recovery of sperm mobility was independent of time when sperm were stored at 10°C for 5 h (P > 0.05). Potassium chloride was replaced with potassium glutamate in the last experiment. Whereas reactivation was once again independent of time when sperm were stored with glucose (P > 0.05), greater variability was observed among observations in comparison with the potassium chloride-based medium. In summary, sperm motility was inactivated when calcium was chelated and extracellular sodium was replaced with potassium. Sperm reactivation was most consistent when chloride was the predominant extracellular anion. Collectively, these experiments demonstrate that short-term sperm storage can be achieved with simple media that promote sperm glycolysis and minimize energy demands imposed by the active transport of calcium and sodium ions.