Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.     

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm.     

Effect of Freezing Rates and Supplementation of α-Tocopherol in the Freezing Extender in Equine Sperm Cryosurvival

This study was conducted to determine the impact of antioxidant (α-tocopherol) supplementation in the cryopreservation extender and three different freezing rates (FRs) on quality of post-thaw semen to elaborate a new protocol for stallion semen cryopreservation. Six ejaculates from each of four stallions were subjected to cryopreservation with a commercial extender (Gent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2 mM α-tocopherol. The semen was exposed to three different freezing rates between 5°C and −15°C: slow (5°C/min), moderate (10°C/min), and fast (20°C/min). After thawing, the sperm viability (Sybr-14 and propidium iodide [PI]), mitochondrial membrane potential (MMP) (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine), lipid membrane peroxidation (LPO; C₁₁-BODIPY^581/591), and apoptosis status (fluorescein isothiocyanate–conjugated annexin V and PI) of the plasmatic membrane of each sample were determined by flow cytometry. For both extenders, the percentage of viable cells was higher for spermatozoa cooled at 5°C/min than at 10°C/min and 20°C/min (P ≤ .05). The FR of 20°C/min demonstrated a lower value of MMP than the FR of 5°C/min and 10°C/min (P ≤ .05). The α-tocopherol extender improved (P ≤ .05) post-thaw membrane LPO; however, it did not improved viability and the apoptosis status of the sperm plasmatic membrane after thawing. In conclusion, results clearly indicate that the cryosurvival of stallion spermatozoa is improved when FR of 5°C/min is used from 5°C to −15°C.     

Extenders modify the seminal plasma ability to minimize freeze‐thaw damage on ram sperm

Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze‐thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk‐based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen‐thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin‐based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze‐thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non‐capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.     

Effects of Cryoprotectant Agents on Equine Ovarian Biopsy Fragments in Preparation for Cryopreservation

The exposure effect of cryoprotectant agents (CPAs) on morphology of preantral follicles (PAFs), stromal cell and PAF densities, and area of equine ovarian fragments were evaluated. Three independent experiments with identical methodologies were performed. Each experiment was composed of one CPA (dimethyl sulfoxide, ethylene glycol, or propylene glycol) and was performed in three replicates. Ovarian biopsy fragments were harvested from six mares in each experiment and submitted to the cryoprotectants using four times of exposure (0, 10, 15, and 20 minutes). PAF and stromal cell densities, and area of the fragments were not affected (P > .05) by any of the CPAs throughout the time of exposure. However, the morphology of the PAFs was affected (P < .05) by the CPAs. In the propylene glycol and dimethyl sulfoxide, higher (P < .05) percentages of abnormal PAFs were observed at 10 and 20 minutes of exposure, respectively. The PAF morphology in the ethylene glycol treatments was not affected (P > .05) throughout the times of exposure. Positive correlations (r = 0.57–0.77; P < .001, power = 96%–99%) were identified between PAF density and stromal cell density in all experiments. In conclusion, (1) ethylene glycol seems to be a less harmful CPA to equine PAFs, (2) exposure to CPAs did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAFs.     

A Comparison of the Ability of Three Commercially Available Diluents to Maintain the Motility of Cold Stored Stallion Semen

The objective of this study was to compare the ability of three commercially available extenders to promote poststorage motility of stallion spermatozoa stored at 5°C with and without centrifugation to remove the seminal plasma. Diluents tested included skim milk glucose (SKMG), INRA 96, and VMD-Z. All diluents were tested with (-SP) and without (+SP) centrifugation to remove most of the seminal plasma. In experiment I, after 48 and 72 hours of storage, total (TM) and progressive (PM) motility values were higher (P ≤.05) for those aliquots subjected to the INRA 96-SP as compared with either SKMG treatment. After 72 hours of storage, PM of spermatozoa stored in VMD-Z-SP was superior to that of spermatozoa stored in SKMG regardless of the presence of seminal plasma (P ≤.05). In the second experiment, after 48 hours of storage, PM of spermatozoa subjected to the INRA 96-SP and VMD-Z-SP treatments were superior (P ≤.05) to those for all treatments that had been stored without removal of seminal plasma. Removal of the seminal plasma and resuspension of the sperm pellet with either INRA 96 or VMD-Z resulted in TM after 48 hours of storage that were similar to those obtained after 24 hours of storage.     

The Combinatorial Effect of Different Equex STM Paste Concentrations,Cryoprotectants and the Straw‐Freezing Methods on the Post‐Thaw Boar Semen Quality

This study was to evaluate the combinatorial effect (14 treatments, A–N) of different Equex STM paste concentrations, cryoprotectants and the straw‐freezing method on the post‐thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post‐thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post‐thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw‐freezing method changes between glycerol and DMA.     

Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant

A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml^‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml^‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml^‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.     

Influence of idebenone on ram semen quality stored at 4°C

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris‐based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer‐assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin‐nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id‐treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.     

Use of Direct Thaw Insemination to Establish Pregnancies with Frozen–Thawed Semen from a Standard Jack

Pregnancy rates reported after artificial insemination with frozen–thawed jack spermatozoa have been relatively low compared with those attained in other species. Cholesterol is known to influence post-thaw fertility of both jack and stallion semen, and altering the amount of cholesterol in the freezing extender may help improve the fertility of frozen–thawed jack semen samples. In this study, we report clinical work that was performed using semen samples collected from a single jack. Samples were extended in EZ Mixin OF and then slowly cooled to 5°C. Extended semen samples were centrifuged at 400 × g for 10 minutes and the supernatant was discarded. Spermatozoa were resuspended in freezing medium to a final concentration of 400 × 10⁶ cells/mL and were later frozen in liquid nitrogen vapor. Freezing extender treatments containing 2% ethylene glycol included the following: (1) 20% egg yolk (EY), (2) 5% EY, and (3) 20% EY + 60 mM hydroxypropyl-β-cyclodextrin (β-CD). For this study, a total of 28 mares aged 2 to 18 years was used over five breeding seasons (82 total cycles). Mares were administered human chorionic gonadotropin to induce ovulation when the dominant follicle was ≥35 mm in diameter. They were inseminated within 6 hours before ovulation and again within 6 hours after ovulation. Pregnancy rates obtained were as follows: (1) 6.25% (one of 15 matings) for 20% EY, (2) 46.5% (20 of 43 matings) for 5% EY, and (3) 58.5% (14 of 24 matings) for 20% EY + 60 mM β-CD. These data suggest that binding of cholesterol with β-CD enhances post-thaw fertility of jack semen samples. We conclude that acceptable pregnancy rates could be achieved with frozen–thawed jack semen samples cryopreserved in 5% EY or 20% EY + 60 mM β-CD using direct post-thaw insemination.     

Use of polyvinyl alcohol as a chemically defined compound in egg yolk‐free extender for dog sperm cryopreservation

The objectives of this study were to investigate the effects of polyvinyl alcohol (PVA) as a chemically defined compound in egg yolk (EY)‐free extender by determining the appropriate concentration of PVA and the effect of pH adjustment in EY‐free PVA extenders on dog spermatozoa. Spermatozoa (1 × 10⁸ cells/ml) were frozen with EY‐free extenders supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3 g/100 ml PVA. Sperm progressive motility (PM) was assessed immediately after thawing (IAT) and post‐thaw incubation (PTI), while viability, acrosome integrity and reactive oxygen species (ROS) levels were evaluated after PTI. Additionally, spermatozoa were frozen using EY‐free PVA extenders before pH adjustment (6.45) and after adjustment of pH (6.85). Viability, PM, ROS and gene expression (BCL2 and SMCP) were assessed. Supplementation with 0.05 g/100 ml or more PVA significantly increased PM compared to the control group in the IAT and PTI. Post‐thaw incubation significantly increased sperm motility in all groups. The acrosome integrity in all PVA groups was higher (p < .05) than the control without an effect on ROS and viability. Adjustment of the pH to 6.85 improved (p < .05) sperm PM compared to the non‐adjusted groups without affecting viability, ROS or expression of BCL2 and SMCP. We suggest that PVA supplementation in EY‐free Tris extenders can effectively protect dog spermatozoa during freezing and can maintain higher motility and acrosome integrity. Adjustment of pH in EY‐free PVA extenders can improve post‐thaw sperm motility. Therefore, PVA can be used as a compound in EY‐free extender for the cryopreservation of dog spermatozoa.     

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk extender

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk (TNM) extender was examined in this study. Pooled semen samples obtained from West African Dwarf (WAD) goat bucks were diluted with Tris‐based extenders containing different levels of TNM (0, 5, 10, 15 and 20 ml/100 ml extender). The diluted semen samples were subjected to slow and rapid freezing for a period of 7 days and thereafter evaluated for sperm functional attributes (percentage motility, acrosome integrity, membrane integrity, abnormality and livability) and oxidative stress (malondialdehyde [MDA] concentration and acrosin activity) parameters. Results showed that higher (p < 0.05) motility, livability, membrane and acrosome integrities in semen cryopreserved with slow freezing compared to rapid freezing. These parameters (motility, livability and membrane integrity) were higher (p < 0.05) in semen cryopreserved with 15% TNM in both slow and rapid freezing protocols. The results revealed that semen cryopreserved in slow freezing had lower (p < 0.05) abnormality compared to rapid freezing. Acrosin activity was higher in slow freezing compared to rapid freezing. Acrosin activity was higher at 15% TNM in both slow and rapid freezing. Lower (p < 0.05) MDA concentration was observed in semen cryopreserved using slow freezing compared to rapid freezing. The findings revealed improved post‐thaw sperm functional attributes and oxidative stress parameters of WAD goat spermatozoa cryopreserved with 15% TNM using slow freezing.     

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration,freezing rate and thawing rate on post-thaw semen quality

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality.

2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN₂) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined.

3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found.

4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN₂ surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.     

Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balaenoptera bonarensis) spermatozoa

The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.     

Freezability of Tushin Ram Semen Extended with Goat or Cow Milk Based Extenders

Cow milk is used as an extender for ram semen cryopreservation. Caseins, the major proteins of milk, appear to provide some protective effect to sperm during cryopreservation. Goat milk has unique casein structure. The aim of this study was to investigate effect of goat milk, as a main semen extender, on freezability of Tushin Ram semen. For this aim, ejaculates from four Tushin rams were collected with artificial vagina and pooled. Pooled semen was separately extended with four different extenders: TRIS based (TRIS), cow skim milk based (CSM) (10 g/100 ml), cow semi‐skim milk based (CSSM) and goat semi‐skim milk based (GSSM) extenders, containing egg yolk and glycerol. The semen was cryopreserved and stored in liquid nitrogen until examination date. After thawing (at 37°C for 1 min), sperm motility, viability, morphology, acrosome and membrane integrity (HOST) were evaluated. Although, there was not any significant differences between extenders in post‐thaw percentage of viable spermatozoa (p > 0.05), Tushin ram semen extended with GSSM or CSM extenders had significantly higher post‐thaw percentage of progressive motility (25.0% and 30.8% respectively), compared with CSSM and TRIS (7.5% and 14.1% respectively, p < 0.001). Moreover, lowest abnormality percentage of post‐thaw spermatozoa were detected in ram semen extended with GSSM (49.5%) and CSM (51.5%), compared with CSSM (65.7%) and TRIS (60.7%) (p < 0.05). Whilst the results were considered, it was concluded that goat milk based extenders may be effectively and trustfully used in cryopreservation of Tushin ram semen, instead of cow milk and Tris based extenders, as a main extender.     

Alternatives in Donkey semen cryopreservation: Mare vs. Jenny Colostrum

The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated the sperm motility by means of computer‐assisted analysis, viability by SYBR‐14 and propidium iodide (PI), membrane functional by HOS test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC‐PNA) and PI. The results demonstrated that lactose–jenny colostrum extender displayed significantly higher values (p < .05) in nearly all parameters evaluated – Total Motility, Viability, HOS test, VCL, VSL, VAP, LIN, STR and WOB –, compared with mare colostrum and egg yolk extenders after thawing. In conclusion, the extender containing jenny colostrum used for donkey semen cryopreservation improved the donkey sperm quality after the freezing–thawing process.     

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.     

Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen

The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at +37°C, centrifuged (600 × g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 × 10⁶ spermatozoa/ml. The resulting mixture was cooled to 4°C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at +4°C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at −196°C and stored. Two straws per extender and per ejaculate were thawed in a water bath at +37°C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at +37°C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender.