Turkey Spermatozoa Research Articles

The effect of diluent, temperature and age on turkey spermatozoa motility in vitro

The aim of this study was to evaluate the turkey spermatozoa motility in in vitro conditions and to prove the effect of different conditions of incubation – diluents, temperature and age of birds. Spermatozoa were obtained from adult turkey's line of Big 6, and spermatozoa motility parameters were evaluated using a computer-assisted semen analyzer (CASA) system. Significant decrease of spermatozoa motility at laboratory temperature (22°C) was detected from time 0 (94.15%) till 180 minutes of incubation (53.91%). At the cool media incubation (5°C), this difference was lower (95.41 and 78.86%, respectively), and the differences were significant from 30 minutes of incubation till 180 minutes. Progressive spermatozoa motility replicated the tendency of total spermatozoa motility. When the physiological solution to commercial diluent at 5°C was compared, the spermatozoa motility and progressive motility in both groups were very consistent for 90 minutes of incubation. Subsequently, significantly higher spermatozoa motility was detected at time periods 120, 150 and 180 minutes of incubation in commercial diluent. Motility was also higher in this group after 24 hours. Influence of age on spermatozoa motility parameters was analysed at 22°C at the time 0 and after 30 minutes of incubation. Analysis of spermatozoa motility as well as progressive spermatozoa motility proved higher values in Group A (aged 35–42 weeks) compared to Group B (aged 63–73 weeks). These results clearly suggest that low temperature and commercial diluents maintain motility parameters during longer time periods and the increasing age of birds has negative impact on motility parameters.

Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2×2×2×5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8–31.5%) of fertile, non-viable embryos (Day 1–6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10–13weeks and 9–10weeks, respectively. The duration of hatchability was 4–6weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5±2.4%), followed by the E line (5.3±1.3%), F line (3.7±2.0%) and RBC2 line (2.6±0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.

Effect of dialysis on the proacrosin/acrosin system and motility of turkey (Meleagris gallopavo) spermatozoa during liquid storage

1. The effect of dialysis on the proacrosin/acrosin system and motility of turkey spermatozoa were examined after 24 and 48 h of liquid storage at 4°C.2. Fifteen pools of semen diluted in extender were dialysed against Clemson Turkey Semen Diluent (dialysed semen) or stored in aerobic conditions (undialysed semen). Semen quality was assessed by measuring spermatozoa motility, amidase activity of spermatozoa suspension, spermatozoa extract and seminal plasma and anti-trypsin activity of seminal plasma.3. Extracted amidase activity of dialysed semen was lower than undialysed by 28%. Higher values for speed parameters of spermatozoa were found in dialysed semen in comparison to undialysed, for example, 81.6 µm/s versus 75.0 µm/s for straight-line velocity (VSL), 114.7 µm/s versus 110.3 µm/s for curvilinear velocity (VCL) and 86.6 µm/s versus 79.8 µm/s for average path velocity (VAP).4. It was concluded that dialysis caused lower amidase activity of spermatozoa and increased speed parameters of progressively motile turkey spermatozoa during storage. Lower extracted amidase activity of dialysed semen reflected better membrane integrity of dialysed semen and suggests that the proacrosin/acrosin system of dialysed spermatozoa is less susceptible to activation compared to undialysed semen.

Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa

1. The effects of lycopene-enriched extenders on the in vitro quality of turkey semen including lipid peroxidation were examined after chilled and frozen storage. 2. Five pools of semen diluted in extenders containing 0, 0·05 or 0·1 mg/ml of lycopene were stored at 5°C for 48 h or cryopreserved as pellets and the following variables determined in fresh samples and samples stored chilled or frozen: sperm motility, viability, osmotic resistance, DNA integrity and lipid peroxidation (as malonaldehyde production). 3. Semen quality was generally compromised after storage, especially post-freezing. However, in the presence of the highest dose of lycopene, both the viability and osmotic-resistance of chilled spermatozoa and the DNA integrity of frozen spermatozoa were similar to those of fresh spermatozoa. 4. Greater lipid peroxidation was detected in refrigerated compared to fresh or cryopreserved spermatozoa. However, spermatozoa chilled in lycopene-enriched extenders showed significantly lower malonaldehyde levels than those chilled without lycopene, while the addition of lycopene to the freezing medium served to maintain the lipid peroxidation levels observed in fresh semen. 5. In conclusion, the presence of lycopene in the extender improved the survival of turkey spermatozoa after liquid-storage and protected DNA integrity against cryodamage. The beneficial effects of lycopene observed could be related to its capacity to diminish sperm lipid peroxidation during refrigeration or cryopreservation.

Use of phosphatidylcholine to improve the function of turkey semen stored at 4°C for 24 hours

The ability to store turkey semen for 24 h in vitro without a significant loss in fertility would benefit the commercial turkey industry. We investigated the use of exogenous phosphatidylcholine to improve the viability, mobility, hydrolyzing ability, and fertility of stored turkey sperm. For experiment 1, semen was diluted 1:1 with extender containing 0.0 (control), 0.5, 2.5, or 10.0 mg/mL of phosphatidylcholine labeled with a fluorochrome and maintained under aerobic conditions for 24 h at 4°C. Semen aliquots were removed at 30-min intervals during the first 4 h and at 1-h intervals from 8 to 24 h of storage for fluorometric evaluation by flow cytometry. Turkey sperm incorporated labeled phosphatidylcholine in a dose-dependent manner during the first 12 h of storage (P<0.05). At 24 h of storage, phosphatidylcholine uptake increased 7.8-fold for the 0.5-mg treatment, 9.2-fold for the 2.5-mg treatment, and 6.7-fold for the 10-mg labeled phosphatidylcholine treatment. For experiment 2, turkey semen was diluted and stored as for experiment 1 except the phosphatidylcholine was unlabeled. After 24 h, the viability, mobility, hydrolyzing ability, and fertility of turkey sperm was assessed. Supplemental phosphatidylcholine did not improve (P>0.05) the viability or mobility of stored sperm. At 2.5 mg/mL, phosphatidylcholine improved the hydrolyzing ability of stored sperm compared with control or other phosphatidylcholine treatments (P<0.05). The mean fertility rate of eggs from hens inseminated with control semen was 33.5%±4.5. Use of 0.5, 2.5, or 10.0 mg phosphatidylcholine/mL improved (P<0.05) the fertility rates during the first 11 wk of egg production; higher fertility rates occurred with 2.5 mg phosphatidylcholine/mL compared with other phosphatidylcholine treatments for 5 of those 11 wk (P<0.05). We conclude that supplemental phosphatidylcholine appears to counteract the damaging effects of lipid peroxidation or enzymatic degradation during in vitro storage by providing exogenous phospholipids for incorporation into the turkey sperm plasma membrane.

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.

Identification of the Second Form of Acrosin in Turkey Spermatozoa

Acrosin from turkey spermatozoa has been recently identified and characterized. In this study, we reported the identification of second form of acrosin (acrosin II) in turkey spermatozoa. Using the three-step isolation procedure, we purified and characterized the acrosin II from a turkey spermatozoa extract. N-terminal Edman sequencing allowed the identification of the 24 amino acids from the internal part of acrosin II: SLQEYVEPYRVLQEAKVQLIDLNL. Thanks to homology alignment, we concluded that acrosin II is an acrosin-like protein similar to avian acrosin, including turkey acrosin. The molecular mass of acrosin II estimated by mass spectrometry was 30.869 kDa. During chromatofocusing, the acrosin II was eluted at pH range from 6.4 to 6.2. Acrosin II was found to be a glycoprotein. The glucosamine and galactosamine were present in carbohydrate structures of acrosin II. Acrosin II is characterized by similar physicochemical characteristics like previously identified bird acrosins, including acrosin from turkey spermatozoa. Similarities between turkey acrosins were also confirmed immunologically by western blot analysis. It can be suggested that two forms of serine proteinase similar to acrosin exist in turkey spermatozoa. These phenomena of both acrosins in spermatozoa agree with the concept of functional redundancy of proteolytic enzymes in the reproductive system. These redundancies may be important for efficient fertilization in turkey.

Cryopreservation of turkey semen by the pellet method: Effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis

This study was designed to identify the best pellet cryopreservation procedure for the cryosurvival of turkey semen among 192 different treatments established by variations and permutations of seven conditions used in the freezing/thawing process. These conditions were: diluent (IGGKPh, SPh or Tselutin); dilution rate (1:3 vs. 1:4); cooling time (45 vs. 60 min); dimethylacetamide (DMA) concentration as cryoprotectant (6 vs. 8%); equilibration time in DMA (1 vs. 5 min); semen drop volume (50 vs. 80 μL) and thawing temperature (60 vs. 75 °C). Through principal components analysis (PCA), post-thaw sperm quality data (mobility, viability and membrane functional integrity) were reduced to a single output variable (Sperm Quality) indicating overall post-thaw semen quality. All treatments induced a significant reduction in semen quality after warming (P < 0.01), though one set of seven conditions, or treatment, was identified by PCA to generate the highest Sperm Quality score and a further five treatments yielded a score not significantly different (P > 0.05) from this best score. Although still not fulfilling the requirements for commercial application, our findings serve to identify the critical steps in turkey sperm cryopreservation that need to be assessed in future studies.

Effect of organic and inorganic forms of selenium in diets on turkey semen quality

The effects of Se supplementation and its organic or inorganic form on semen quantitative parameters (ejaculate volume, sperm concentration, and total number of sperm) and biochemical parameters of seminal plasma (protein concentration, acid phosphatase activity, superoxide dismutase activity, and total antioxidant capacity) were investigated over a 25-wk reproductive season. Additionally, DNA fragmentation and motility characteristics of turkey spermatozoa were measured. The parameters of turkey semen in relation to yellow semen syndrome were also determined. Twenty-four males (Big 6) were divided into 3 experimental groups differing in form of Se supplementation (no Se supplementation, 0.3 mg/kg of inorganic Se from sodium selenite and 0.3 mg/kg of organic Se from Sel-Plex, Alltech Inc., Nicholasville, KY). Dietary Se supplementation enhanced the sperm concentration and total number of sperm and did not influence the antioxidative properties of turkey seminal plasma and most biochemical parameters. Only seminal plasma acid phosphatase activity was increased in turkeys fed inorganic Se. The main sperm DNA fragmentation parameters were not affected by dietary Se. The highest percentage of motile spermatozoa (85%) was recorded for the semen of turkeys fed organic Se. Values of the biochemical parameters (acid phosphatase, superoxide dismutase, total antioxidant capacity) of seminal plasma increased during the reproductive season. Yellow semen was characterized by increased biochemical parameters and decreased spermatozoa motility characteristics. However, the percentage of motile spermatozoa did not differ between white and yellow semen. Organic Se seemed to be the preferred form of diet supplementation in comparison with inorganic Se. Biochemical parameters of semen and spermatozoa motility parameters appear to be useful for evaluating the effect of age on semen quality. Monitoring the DNA fragmentation of spermatozoa at the end of the reproductive season could be a useful tool for monitoring turkey semen quality. Increased superoxide dismutase activity can be used as an indicator of yellow semen. A decline in the quality of yellow semen can be related to a decrease in the spermatozoa motility parameters of turkeys.

Comparative cryopreservation of avian spermatozoa: Benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6–26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin–eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

Effect of different sperm concentrations on the post-thaw viability and motility of turkey spermatozoa cryopreservedby the pellet method

The effects of different prefreezing semen concentrations on post-thawing quality of turkey semen cryopreserved with the pellet method were investigated. Ten pooled semen samples were each divided into 6 subsamples and diluted with Tselutin extender to obtain a final concentration of 0.5, 1, 2, 3, 4 and 5x109/mL respectively. Subsamples were cooled, added with 8% of dimethylacetamide as cryoprotectant and, after 5 minutes of equilibration, seminal aliquots of 80 μL were directly dropped into a liquid nitrogen bath to form frozen pellets. Thawing was performed in few seconds at 75°C. Sperm motility (Accudenz® swim-down test), viability (SyBr-Propidium Iodide staining) and sperm susceptibility to osmotic stress (Hyposmotic-water test) were assessed. Cryopreservation caused an overall loss of sperm quality, however differences in seminal parameters due to the different sperm concentration were observed in turkey spermatozoa after thawing: spermatozoa diluted to 4x109/mL showed significant higher values in mobility, viable and osmotic resistant spermatozoa compared to the other concentrations. This study showed that the post-thaw quality of turkey semen cryopreserved by pellets method was affected differently in relation to prefreezing sperm concentration.

Sperm motility enhancement with low level laser therapy

OBJECTIVE: To quantify the effects of Low Level Laser Therapy (LLLT) on human sperm motility in-vitro. Significant literature details the photo-bio-modulation effect of LLLT on human tissue from both laser and light-emitting diode (LED) sources at wavelengths from 630 to 1000nm but no data exists on spermatozoa. The intracellular effects arise predominantly from stimulation of ATP production via the respiratory chain of the mitochondria. LLLT has been shown to temporarily improve canine sperm motility, increase calcium transport within murine spermatozoa, increase the number of acrosome reacted bull sperm, and increase the longevity of motility in frozen turkey sperm.DESIGN: Prospective observational study on normospermic and asthenospermic semen specimens.MATERIALS AND METHODS: Four human semen specimens were subjected to LLLT from two continuous output sources at varying distances using the Thor-Laser system: a 104 LED diode cluster of 660nm and 850nm (100-400 Joule dose) and a 200mW laser 810nm GaAlAr diode (2-4 Joule dose). Sperm motility of both test and control aliquots was assessed using the sperm motility index (SMI) and total functional sperm count (TFSC) parameters measured on a SQA IIB analyser.RESULTS: The bio-modulation from both sources was consistent across samples with different degrees of effect. The SMI and TFST increased up to four fold for normal semen and seven fold for asthenospermic semen compared to controls, with an inhibitory effect at higher doses. The maximum effect and timing post-exposure varied with the light source. For normal semen it was at the lowest dose at 30 minutes for the LED cluster and at the intermediate doses at 15 minutes for the diode laser. For asthenospermic semen it came from the mid-range doses at 30 minutes for the LED cluster and from the lowest dose for laser at 30 minutes post-exposure.CONCLUSIONS: Human sperm motility is modified by exposure to LLLT with an effect that is both dose and sample dependent. This justifies further study of varying semen quality, doses and source types of light, and verification that it is not detrimental to DNA integrity prior to evaluation of its effect on fertilizing capacity. OBJECTIVE: To quantify the effects of Low Level Laser Therapy (LLLT) on human sperm motility in-vitro. Significant literature details the photo-bio-modulation effect of LLLT on human tissue from both laser and light-emitting diode (LED) sources at wavelengths from 630 to 1000nm but no data exists on spermatozoa. The intracellular effects arise predominantly from stimulation of ATP production via the respiratory chain of the mitochondria. LLLT has been shown to temporarily improve canine sperm motility, increase calcium transport within murine spermatozoa, increase the number of acrosome reacted bull sperm, and increase the longevity of motility in frozen turkey sperm. DESIGN: Prospective observational study on normospermic and asthenospermic semen specimens. MATERIALS AND METHODS: Four human semen specimens were subjected to LLLT from two continuous output sources at varying distances using the Thor-Laser system: a 104 LED diode cluster of 660nm and 850nm (100-400 Joule dose) and a 200mW laser 810nm GaAlAr diode (2-4 Joule dose). Sperm motility of both test and control aliquots was assessed using the sperm motility index (SMI) and total functional sperm count (TFSC) parameters measured on a SQA IIB analyser. RESULTS: The bio-modulation from both sources was consistent across samples with different degrees of effect. The SMI and TFST increased up to four fold for normal semen and seven fold for asthenospermic semen compared to controls, with an inhibitory effect at higher doses. The maximum effect and timing post-exposure varied with the light source. For normal semen it was at the lowest dose at 30 minutes for the LED cluster and at the intermediate doses at 15 minutes for the diode laser. For asthenospermic semen it came from the mid-range doses at 30 minutes for the LED cluster and from the lowest dose for laser at 30 minutes post-exposure. CONCLUSIONS: Human sperm motility is modified by exposure to LLLT with an effect that is both dose and sample dependent. This justifies further study of varying semen quality, doses and source types of light, and verification that it is not detrimental to DNA integrity prior to evaluation of its effect on fertilizing capacity.

Characterizing the Glycocalyx of Poultry Spermatozoa: II. In Vitro Storage of Turkey Semen and Mobility Phenotype Affects the Carbohydrate Component of Sperm Membrane Glycoconjugates

The turkey sperm glycocalyx is known to contain residues of sialic acid, alpha-mannose/alpha-glucose, alpha- and beta-galactose, alpha-fucose, alpha- and beta-N-acetyl-galactosamine, monomers and dimers of N-acetyl-glucosamine, and N-acetyl-lactosamine. Potential changes in these carbohydrates during in vitro semen storage at 4 degrees C were evaluated using males of both high- and low-sperm-mobility phenotypes. Changes in carbohydrate residues were quantified by flow cytometry analysis using a battery of 14 fluorescein isothiocyanate-labeled lectins in combination with control (sialylated) or neuraminidase-treated (nonsialylated) sperm. Sperm were evaluated at 0, 2, 4, 8, 12, and 24 hours of storage. For control sperm, 4 different patterns of lectin binding were observed over time: 1) increased mean fluorescence intensity (MnFI) at 2 hours (Griffonia simplicifolia lectin-I [GS-I]) and 8 hours (Ricinus communis lectin-I [RCA-I]) that remained elevated during storage; 2) increased MnFI at specific time points (Limax flavus lectin [LFA], 2 hours; Artocarpus integrifolia lectin [jacalin] and succinyl Triticum vulgare lectin [sWGA], 8 hours; Galanthus nivalis lectin [GNA], 12 hours) followed by decreasing MnFI during the remainder of the 24-hour storage period; 3) increased MnFI only at the 24-hour time point (Lotus tetragonolobus lectin [lotus] and Arachis hypogaea lectin [PNA]); and 4) no changes in MnFI during the 24-hour storage period (Erythrina cristagalli lectin [ECA], GS-II, Pisum sativum lectin [PSA], Glycine max lectin [SBA], and Wisteria floribunda lectin [WFA]). For nonsialylated sperm, increased binding of ECA, GS-II, SBA, and WFA was observed at variable time points; only Canavalia ensiformis lectin (Con A) and PSA remained unchanged during storage. Differences between mobility phenotypes existed for lectins Con A, GS-II, LFA, PSA, SBA, and sWGA, with sperm from low-mobility males exhibiting higher MnFI than high-mobility males throughout 24 hours of storage. We concluded that the observed increases in lectin binding during semen storage indicate an augmentation of nonsialylated terminal residues, which could alter sperm antigenicity and negatively impact fertility. Further, spermatozoa from low-mobility males may have higher antigenicity even before semen storage. Other possible functional implications are discussed.

Liquid storage of turkey semen: Changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa

The study considered two major aims: (a) to measure the changes in quality parameters, lipid composition and antioxidant activity occurring in turkey spermatozoa during liquid storage; (b) to determine if the enrichment of sperm in n-3 fatty acids and alpha-tocopherol affect sperm survival during storage. Turkey breeders were fed a control diet or an Omega3 diet enriched with fish oil and alpha-tocopheryl-acetate. Ejaculates were pooled (5 ejaculates/pool; 4 pools/treatment) and stored in vitro for 48 h at 4 °C. Viability, motility, susceptibility to induced peroxidation and alpha-tocopherol content were measured in spermatozoa; lipid and phospholipid fatty acid composition were measured in spermatozoa and seminal plasma. The proportion of motile and viable spermatozoa significantly decreased, and the proportion of dead spermatozoa significantly increased. The susceptibility of turkey spermatozoa to induced peroxidation also significantly increased during storage. The enrichment of turkey spermatozoa with n-3 long chain PUFA and vitamin E by dietary treatment did not prevent the negative effect of storage on sperm quality and sensitivity to induced in vitro peroxidation; however, it was efficient in partially prevent the increase of sperm death, therefore the proportion of dead spermatozoa was higher in control (37.4%) compared to treated spermatozoa (31.7%) after 48 h liquid storage. Major changes were recorded in the lipid composition of turkey spermatozoa during liquid storage in both experimental dietary groups, whereas no significant changes were measured in seminal plasma. In spermatozoa, a great loss in the phospholipid and free cholesterol content was measured. Moreover, the loss in total sperm phospholipid was associated to a peculiar and selective decrease in the bounded fatty acids: saturates and monounsaturates were greatly reduced and polyunsaturates did not change. As a consequence, the polyunsaturated to saturated fatty acid ratio increased during 48 h liquid storage. The observed changes in the lipid and phospholipid-bound fatty acid composition of turkey spermatozoa occurring during liquid storage might be related to different events and have been discussed.

Turkey Sperm Reside in the Tubular Glands in the Urodeum Following Artificial Insemination

A turkey hen in egg production requires 48 h after the last insemination to maximize the number of sperm in the uterovaginal junction sperm-storage tubules. Where the sperm that continue to fill the oviductal sperm-storage sites during this 48-h period reside remains unknown. Histological sections of the juncture of the vagina with the urodeum, the central compartment of the cloaca, revealed deep tubular glands containing periodic acid-Schiff-positive secretory material. When examined 36 h after the last artificial insemination, sperm were observed in the lumen of the tubular glands associated with the urodeum. We suggest that sperm reside in the tubular glands within the urodeum and are released in association with the secretory activity of the tubular glands. These sperm then may ascend the vagina to continue to populate the sperm-storage tubules. Alternatively, the sperm in the tubular glands of the urodeum may be evidence of spermatorrhea and have no functional role in the fertilization process.

Osmotic tolerance of avian spermatozoa: Influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane ( Grus canadensis) and turkey ( Meleagridis gallopavo). Sperm osmotolerance (300–3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 °C versus 21 °C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me 2SO) at either 4 °C or 21 °C; and (3) inhibition of the Na +/K + and the Na +/H + antiporter membrane ionic pumps. Sperm viability was assessed using the eosin–nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 °C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na +/K + and the Na +/H + antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.

Characterizing the Glycocalyx of Poultry Spermatozoa: I. Identification and Distribution of Carbohydrate Residues Using Flow Cytometry and Epifluorescence Microscopy

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.

Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa

Short-term liquid storage of turkey semen is of great interest in the management of turkey reproduction due to the extensive use of artificial insemination. This study examined changes in DNA fragmentation (using a comet assay), sperm motility characteristics (using computer-aided sperm analysis), and amidase activity (using a colorimetric assay) of turkey sperm stored for 24 and 48 h. In addition we found that turkey spermatozoa contain besides acrosin, additional two serine proteinases of molecular weight of 34 and 42 kDa. We found that, after 48 h of liquid storage, decreases in sperm motility characteristics and increases in amidase activity and DNA fragmentation occurred. An increase of amidase activity was found after 24 h. Decreases in sperm motility and increase in DNA fragmentation were found after 48 h of storage. These data suggest that a decrease in turkey sperm quality during short-term storage is related to disturbances to the acrosome, presumably related to premature activation of acrosomal serine proteinases, and to a lesser extent a decrease in sperm motility characteristics and damage of sperm DNA.

Effect of Semen Extender and Density Gradient Centrifugation on the Motility and Fertility of Turkey Spermatozoa

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.