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The experiment was conducted to evaluate the metabolic activity through MTT (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) reduction assay of Indian red jungle fowl sperm during cryopreservation process. In addition, MTT test was compared with sperm motility and viability data. Sperm analysis was made at pre-dilution, post-dilution, post-cooling, post-equilibration and post freeze-thawing stages. Pearson’s correlation coefficient showed significant (P<0.01) positive association of MTT reduction rate with motility (r=0.85) and viability (r=0.84). A significant (P<0.05) decline was observed in motility, viability and metabolic status after eac h stage of cryopreservation. The highest (P<0.05) stage-specific percent decrease in motility and viability were observed after freeze-thawing. Nevertheless, stage-specific percent decrease in metabolic status differ (P<0.05) after dilution (5.93 ± 0.12), cooling (8.41 ± 0.03), equilibration (19.1 ± 0.04) and freeze-thawing (30.9 ± 0.08). The total decrease in motility, viability and metabolic activity after the complete freeze-thawing cycle were calculated as 56%, 47% and 52%, respectively. It is concluded that MTT reduction assay is simple and reliable method to evaluate mitochondrial redox activity in sperm of this threatened species.

A classical chicken semen diluent (Lake's 7.1 diluent) was modified to have lowered osmolalities (ranging from 290 to 410 mOsm/kg). The modified medium with physiological osmolality of 325 mOsm/kg allowed cold storage of fresh semen for several days with very little loss of membrane integrity and motility, while high osmolalities inhibited motility. This modified medium was then used as base for freezing medium to test effects of the type and concentration of cryoprotective agent (CPA), and the cooling rate (CR). A number of CPAs (methylformamide, methylacetamide, dimethylformamide (DMF), dimethylacetamide (DMA), diethylformamide, and propylene glycol) were first compared by freezing semen with 0.6 mol/l of the respective CPA at a cooling rate of 250 °C/min. Post-thaw motility and membrane integrity were highest with DMA and DMF. Finally, in more detailed factorial experiments, semen from individual cocks or pooled semen was frozen using CRs of 4, 50, 250, and 440 °C/min and DMA concentrations ([DMA]) of 0.4, 0.6, 1.0, and 1.5 mol/l. Straws from each semen sample x treatment combination were divided for semen assessment at three different research groups for sperm motility, membrane integrity, kinked tails, and DNA fragmentation, using microscopy, computer assisted motility analysis, and flow cytometry. There were clear effects of both CR and [DMA] and their interaction. CRs 50 and 250 °C/min gave best post-thaw sperm performance. Higher DMA concentrations gave better post-thaw membrane integrity, but concentrations above 1.0 mol/l can decrease sperm velocity or even inhibit sperm motility. Therefore [DMA] may best be 0.6-1.0 mol/l at a CR of 50-250 °C/min.

Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank., r

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.

The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P.

BACKGROUND Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. RESULTS RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects. CONCLUSIONS Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos

This review provides an updated overview of the seminal plasma composition, and the role of metabolic and protein components on the sperm function of avian species. In addition, the implication of seminal plasma on assisted reproductive techniques of birds was discussed. The semen of birds usually has exceptionally high sperm concentration with relatively little seminal plasma, but this contributes to very fast changes in sperm metabolism and function. The biochemical characteristics and physiological roles of the various seminal plasma components in birds (carbohydrates, lipids, amino acids, hormones, and proteins) are poorly understood. Seminal plasma content of proteins has an action on most cellular functions: metabolism, immunity, oxido-reduction regulation, proteolysis, apoptosis, ion homeostasis, and antimicrobial defenses. The variable amount of many proteins is related to a different fertility capacity of poultry sperm. The role of seminal plasma on semen conservation (chilling and freezing) remains largely a matter of speculation, as both inhibitory and stimulating effects have been found. Whereas the presence of seminal plasma did not seem to affect the sperm survival after freezing-thawing, DNA fragmentation is lower in the absence of seminal plasma. The molecular basis of the influence of seminal plasma on sperm cryo-resistance was also discussed in the present review.

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.

Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P = 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% ± 12.1) than VF cryopreserved sperm control (16.1% + 6.2). Supplementing valine to the freezing extender doubled (P = 0.026) the fertility rate of VF (32.6% ± 12.2) compared wit

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the v

Density-gradients centrifugation (DGC) and filtration columns (FC) are used to separate deformed or dead sperm, debris, and other cells that may negatively affect the fertilizing capacity of sperm in fresh, chilled and frozen/thawed semen. The present study was conducted to evaluate the suitability of DGC (BoviPure®, Percoll® and Accudenz®) and FC (Sephadex G-15®) sperm selection procedures for fresh-extended and cold-stored ram semen by assessment of post-treatment sperm quality variables. Twenty normospermic ejaculates from ten adult Merino rams were used. Sperm concentration of recovered cells was greater (P < 0.001) after BoviPure treatment than other procedures in both fresh and cold semen. With the Sephadex method, there were more desirable values than with use of DGC procedures in several sperm motility variables measured by using the CASA system. In non-refrigerated semen samples, the percentage of progressive sperm motility (%PSM) after Sephadex filtration was greater (P < 0.05) than after BoviPure treatment; the straightline velocity (VSL) value after Sephadex filtration was greater (P < 0.01) than after Accudenz treatment; the amplitude of lateral head displacement (ALH) after Sephadex and Accudenz treatment was less than non-filtered semen (P < 0.001) and after Percoll (P < 0.01) and BoviPure (P < 0.05) treatments. In cold-stored semen samples, the %PSM after Sephadex filtration was greater than non-filtered (P < 0.05) semen and after BoviPure (P < 0.05), Percoll (P < 0.05) and Accudenz (P < 0.001) treatments. It is concluded that Sephadex column filtration can be used to select ram sperm in non-refrigerated and cooled semen, because percentage progressively motile sperm and some other sperm motility characteristics are greater with use of this techniques as compared with use of DGC methods.

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.