OptiXcell Extender Research Articles

Sperm collection in rhinoceros via urethral catheterization

Assisted reproductive technologies are playing an ever-increasing role in the management of ex situ rhinoceros populations. Traditionally, sperm collection in rhinos is conducted via electroejaculation (EEJ) or gamete rescue. Sperm collection via urethral catheterization (UC) is gaining popularity in wildlife reproductive sciences as it requires less specialized equipment, is faster, and less stimulatory than EEJ. This study investigated the use of UC to collect sperm from Southern white (Ceratotherium simum simum; n = 3) and Eastern black (Diceros bicornis michaeli; n = 6) rhinoceros as an alternative or complementary to collection via EEJ. UC samples (n = 14) exhibited high sperm concentration (1.6 ± 0.49 ×109 sperm/mL) and low volume (0.98 ± 0.26 mL). Samples obtained via EEJ (n = 11) were of moderate sperm concentration (0.14 ± 0.05 ×109 sperm/mL) and high volume (30.6 ± 7.2 mL). UC samples were processed for cryopreservation in OptiXcell extender. Motility of UC sperm decreased post-thaw (56 ± 2.4%) over pre-freeze (77.1 ± 3.2%; P < 0.05), with longevity data consistent to prior results published for sperm collected via EEJ. These results suggest sperm quality and cryo-survival are not impacted by the collection method. Since EEJ was conducted after UC, sperm parameters could not be compared between the two methods. Results indicate that UC is a viable alternative to EEJ for sperm collection in rhinoceros.

Association between levels of insulin-like growth factor-1 in serum and seminal plasma with fresh and frozen-thawed semen characteristics in Beetal bucks.

The aim of this study was to investigate the relationship between the levels of insulin-like growth factor-1 (IGF-1) in serum and seminal plasma and the characteristics of semen in Beetal bucks (Capra hircus). A total of 12 adult Beetal bucks were involved in the study, with each buck providing six ejaculates collected using a standard artificial vagina (n = 72 total). Only qualified semen samples (volume of 0.7 mL, a mass motility rating of 3+ or higher on a 0-+ scale, and individual progressive motility of 80% or more) divided into three fractions were processed for estimation of IGF-1 and other seminal parameters like motility, viability, acrosome integrity, sperm abnormality and superoxide dismutase (SOD) activity. The first and second fraction were diluted and extended with Optixcell extender (1:15 ratio). The first ejaculate fraction was processed for studying fresh semen parameters and the second fraction was cryopreserved for evaluating frozen semen parameters. French mini straws (0.25 mL) were used for semen filling, and polyvinyl alcohol powder of different colours was used for sealing the extended semen. The third fraction of each ejaculate was centrifuged at room temperature (1100 × g for 7 min) to separate the seminal plasma. Additionally, blood samples were taken from each buck on the same day as semen collection, resulting in a total of 36 blood samples. The results revealed a significant positive correlation (r = .4243; p < .05) between the concentration of IGF-1 in both serum and seminal plasma of the Beetal bucks. Furthermore, the concentration of IGF-1 in serum showed significant positive correlations with sperm viability (r = .554; p < .05), acrosome integrity (r = .527; p < .05), post-thaw sperm motility (r = .407; p < .01), post-thaw sperm viability (r = .426; p < .01) and post-thaw acrosome integrity (r = .333; p < .05). However, it had a significant negative correlation with SOD activity in fresh semen (r = -0.458; p < .01). Moreover, the concentration of IGF-1 in seminal plasma demonstrated significant positive correlations with individual progressive motility (r = .341; p < .05), sperm viability (r = .527; p < .05), acrosome integrity (r = .539; p < .05), sperm plasma membrane integrity (r = .464; p < .05), post-thaw sperm motility (r = .644; p < .01), post-thaw sperm viability (r = .643; p < .01), post-thaw acrosome integrity (r = .487; p < .01) and post-thaw sperm plasma membrane integrity (r = .521; p < .01). Additionally, it showed a significant negative correlation with SOD activity in both fresh semen (r = -0.714; p < .01) and frozen semen (r = -0.558; p < .01) of Beetal bucks. Based on these findings, IGF-1 in seminal plasma can be considered as a potential biomarker for the selection of bucks for breeding purposes.

Pre-freezing selection of Holstein bull semen with the BoviPure colloid as double- or single-layer centrifugation improves the post-thawing quality

Artificial insemination (AI) is critical for breeding in the dairy industry. High-merit bulls can present low freezability, hampering genetic dissemination. Thawed semen can be improved using density gradient centrifugation (DGC) with colloids, but little information deals with the pre-freezing application. Thus, the BoviPure colloid (optimized for bull spermatozoa) was tested for pre-freezing application as the usual double-layer (DLC) versus single-layer (SLC, quick and economical). Semen from twelve Holstein-Friesian bulls was extended with OPTIXcell extender, frozen (Control), or processed by SLC or DLC and frozen. Sperm were assessed pre-freezing for motility and viability and post-thawing (directly and after 4 h 38 °C) for apoptosis, capacitation status, acrosomal damage, mitochondrial activity, cytoplasmic and mitochondrial reactive oxygen species (ROS), and chromatin status (SCSA for DNA fragmentation and chromatin compaction and monobromobimane, mBBr, for disulfide bridges evaluation). The DGC improved parameters post-thawing (e.g., 57.5%±10.1 motility vs. control 53.3% ± 11.2) at the cost of sperm loss (sperm recovery of DGC 14.4% ± 2.5 and SLC 17.4% ± 2.5). DNA fragmentation (%DFI) decreased (0.21% ± 0.53 vs. control 1.30% ± 0.10), and SLC reduced chromatin compaction. A clustering procedure separated lesser (LF) and greater freezability (GF) bulls. LF samples were especially benefited by DGC, with SLC providing better post-thawing results for this group. In conclusion, pre-freezing DGC improved sperm parameters post-thawing, potentially improving the cryopreservation of low-freezability semen from high-merit bulls. SLC, quicker and economical, would be preferable since it showed similar or higher performance than DLC.

Extension of the equilibration period up to 24 h maintains the post-thawing quality of Holstein bull semen frozen with OPTIXcell®

Semen cryopreservation in bovine livestock is well established, but logistics often require deviations from standard protocols. Extending the equilibration time to the following day is convenient in many situations. To improve our knowledge of the effects of this modification, we studied the post-thawing and post-incubation (4 h, 38 °C) sperm quality after freezing with 4 or 24-h extension in the OPTIXcell extender by using an ample panel of analyses: CASA for motility; flow cytometry for viability, physiology, oxidative stress, and chromatin parameters (DNA fragmentation, chromatin compaction, and thiol groups status); and spectrometry for malondialdehyde production. Semen was obtained from 12 Holstein bulls. The 24-h equilibration time showed few significant effects, with only a tiny decrease in progressive motility and a positive impact on chromatin structure. The incubation removed some of these effects, with the pattern for chromatin compaction remaining the same. No detrimental oxidative stress or increase in apoptotic or capacitation markers was detected. Additionally, the individual bull interacted with the effects of the incubation and the equilibration, especially regarding the chromatin status. Whereas this interaction did not critically affect sperm quality, it could be relevant in practice. Bull fertility as non-return rates (NRR56) was associated with some sperm parameters (especially with an improved chromatin structure) but not in the 4-h post-thawing analysis. Our study supports that extending the equilibration time by at least 24-h is feasible for bull semen freezing with the OPTIXcell extender.

Effect of Quercetin Supplementation in Extender on Sperm Kinematics, Extracellular Enzymes Release, and Oxidative Stress of Egyptian Buffalo Bulls Frozen-Thawed Semen.

Buffalo spermatozoa are more sensitive for cryopreservation compared to other species. This study aimed to evaluate the consequences of quercetin against cryodamage of buffalo frozen–thawed spermatozoa characteristics. Semen of Egyptian bulls (n = 4) was extended in OptiXcell extender incorporated with quercetin at 0 (control), 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 μM before cryopreservation. Frozen–thawed semen was evaluated for sperm motility by computer-assisted sperm analyzer (CASA), viability, morphology, membrane, and acrosome integrities. The kinematics parameters including average path velocity (VAP; μm/s), straight linear velocity (VSL; μm/s), curvilinear velocity (VCL; μm/s), amplitude of lateral head displacement (ALH; μm), beat cross frequency (BCF; Hz), linearity [LIN, (VSL/VCL) × 100], and straightness [STR, (VSL/VAP) × 100] were assessed. The sperm-free extender was evaluated for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and H2O2. Homogenized sperm cells were evaluated for oxidative stress biomarkers [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX)], and lipid peroxidation [malondialdehyde (MDA)]. The highest values of total motility, progressive motility, viability, intact acrosome, and membrane integrity substantially improved with 10 μM of quercetin. STR (%) was substantially low (P < 0.01), and VCL (μm/s) and ALH (μm) were markedly high (P < 0.05) in 10 μM of quercetin. The outflow of ALT enzyme to extracellular fluid was lower with 10 μM of quercetin (P < 0.001) and higher at 2.5 μM of quercetin. The spermatozoa leaked AST was markedly lower at 5.0, 10 (P < 0.001) and 20 μM (P < 0.05) of quercetin. The activity of antioxidant enzymes was eminently low at all quercetin concentrations, and this was accompanied by the decrease in H2O2 in the media. SOD activity at 10–80 μM, CAT at 5.0–40 μM, and GPX at 2.5–80.0 μM of quercetin in spermatozoa were substantially low. MDA level significantly (P < 0.001) decreased at all quercetin concentrations. In conclusion, the incorporation of quercetin at the level of 10 μM is promising in improving buffalo semen characteristics and lower the freezing–thawing oxidative stress.

‘Semen dilution effect’ on sperm variables and conception rate in buffalo

The study aim was to determine how to minimize effects of buffalo semen dilution by evaluating the use of egg yolk (EY), low-density lipoprotein (LDL), and OptiXcell (OC) extenders. Ejaculates (n = 18 from six bulls) were divided into three aliquots that were diluted separately with EY, LDL, and OC extenders corresponding to 20 million (M), 12 M, and 2 M sperm/dose, respectively, and cryopreserved. There were a lesser sperm motility, plasma membrane integrity, and percentage un-capacitated sperm with the 2 M sperm/dose, however, the LDL extender was more effective than OC and EY extender for cryopreservation of buffalo sperm. Excess semen dilution resulted in sustained sperm velocities (curvilinear velocity, average path velocity, and straight linear velocity), and these were greater with use of the OC than LDL and EY extenders. There was no change in amplitude of sperm lateral head displacement (ALH) with respect to dilution, but with regard to extender effects, ALH was greater in sperm extended in LDL and OC than EY. Semen dilution to 2 M sperm/dose resulted in a greater mitochondrial superoxide production. Conception rate (CR) was unaffected with 20 and 12, however, with the 2 M sperm/dose dilutions there was a lesser CR. In conclusion, buffalo semen dilution to the extent of 12 M sperm/dose did not affect most of the seminal variables and CR. Using LDL extender at 2 M sperm/dose protected sperm from the ‘semen dilution effect’ to a greater extent than with use of EY and OC extenders.

Variation in post-thaw sperm quality of white-tailed deer bucks (Odocoileus virginianus) during rut

White-tailed deer farming is a growing industry in the United States, with breeding operations contributing significantly to the industry’s economic impact. Artificial insemination with frozen semen allows for selection and dissemination of valuable genetics, yet surprisingly little is known regarding the best time throughout rut to perform semen cryopreservation. The objective of this study was to compare semen quality following cryopreservation of white-tailed deer bucks collected early in the breeding season (September, n = 6), at peak rut (December, n = 8), and late season (March, n = 7). We hypothesized that post-thaw semen quality would be enhanced at peak rut. Mature bucks were anesthetized with tiletamine-zolazepam and xylazine administered intramuscularly via projector. Semen was collected by electroejaculation and cryopreserved using Optixcell extender. Overall and progressive sperm motilities were assessed for each sample before and after cryopreservation using a computer-aided sperm analyzer. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and DNA stability (acridine orange). Analysis of variance was applied to normalized data using a general linear mixed model with buck ID as a random variable, and a Tukey HSD test was performed as needed for post-hoc analysis. Pre-freeze overall and progressive sperm motilities were lowest in March, intermediate in September, and highest in December (p ≤ 0.04). Post-thaw overall and progressive motilities were lowest in September (p ≤ 0.02), but did not differ between December and March (p ≥ 0.12). The DNA Fragmentation Index was lowest in December, intermediate in September, and highest in March (p ≤ 0.05). The percentage of spermatozoa with intact plasma membrane was higher in December than September (p < 0.01), but the percentage of intact acrosomes per sperm with intact plasma membrane was highest in September (p = 0.03). This study confirms that post-thaw semen quality appears to be superior during peak rut (December) in bucks. Though semen collected early or late in rut may present acceptable motility, DNA stability is impaired, which could adversely affect fertility rates. This data suggests that semen cryopreservation during transitional periods should be avoided, though field studies evaluating the translation of these results into satisfactory pregnancy rates are warranted.

Efficacy of egg yolk based and egg yolk free soya bean milk based extenders for cryopreservation of Zebu cattle and buffalo semen

This study was undertaken on three mature bulls each of Gir cattle and Surti buffalo breeds to evaluate the comparative efficacy of egg yolk based standard TFYG (Tris-citrate-fructose-yolk-glycerol) extender and egg yolk free soybean based commercial extenders Optixcell® (IMV, France) and Andromed® (Minitube, Germany) under split-sample technique. The ejaculates (9/bull) were extended @ 100×106 sperm ml-1 with three extenders and frozen using biofreezer following 4 hr of equilibration. The pooled means of progressively motile sperm observed (irrespective of extenders) at initial, pre-freeze and post-thaw stage in Gir bulls semen were 76.53±0.53, 71.11±0.53 and 39.86±0.90% and in Surti buffalo 80.76±0.39, 74.65±0.45 and 40.35±1.07%, respectively. The corresponding values for live sperm were 75.64±0.76, 69.01±0.97 and 47.99±1.11 % for Gir and 80.90±0.45, 75.76±0.48 and 52.33±0.86 % for Surti buffalo; and those of intact acrosome 94.29±0.25, 90.29±0.27 and 79.29±0.33 % for Gir bulls, and 93.94±0.21, 89.94±0.23 and 78.95±0.26 % for Surti buffalo semen, respectively. The HOS reactive sperm at initial, pre-freeze and post-thaw stage were 76.18±0.74, 71.04±0.76 and 27.90±0.70 % for Gir, and 81.83±0.35, 76.47±0.39 and 27.83±0.68 % for Surti bulls, respectively. The overall mean post-thaw incubation (37°C) survival of spermatozoa observed at 60, 120 and 180 min were 28.40±0.91, 17.78±0.86 and 9.44±0.72% for Gir bulls semen, and 28.01±0.99, 18.40±1.01 and 10.51±0.93% for Surti buffalo semen, respectively. Optixcell was proved superior, and at par with TFYG, than the Andromed in maintaining greater motility, viability, morphology, acrosomal/plasma membrane integrity including post-thaw sperm longevity of cattle and buffalo spermatozoa with significant differences only in sperm motility and post-thaw longevity. The motile, live and HOST reactive sperm were significantly higher in buffalo semen than cattle at initial and pre-freeze stage, but not at post-thaw stage. The results showed that egg yolk free commercial Optixcell extender and egg yolk based TFYG extender were at par in terms of most of the sperm quality traits, hence any one of them can be preferred over Andromed for successful routine cryopreservation of cattle and buffalo semen.

Evaluation of comparative efficacy of egg yolk and soya based extendersfor refrigeration preservation (5ºC) of buffalo semen

The aim of this study was to compare commercially available soybean based extenders, viz. Bioxcell® and Optixcell® (IMV, France) with standard Tris-citrate-fructose-egg yolk-glycerol (TFYG) extender for refrigeration preservation of buffalo semen. Forty eight good quality ejaculates (8/bull) having initial motility &gt;70% obtained at weekly interval in AV from 6 mature Surti buffalo bulls were split-diluted (at 34°C keeping 100×106 sperm ml-1) in TFYG, Bioxcell and Optixcell extenders and stored in refrigerator at 5°C for 72 hrs. The mean percentages of progressively motile spermatozoa observed on dilution (0-hr), and after 24, 48, 72 hrs of refrigeration preservation of semen at 5°C were 78.54±0.30, 68.30±0.30, 59.38±0.35 and 51.63±0.47; live sperm 90.48±0.19, 76.89± 0.51, 69.51±0.35 and 61.86±0.45; morphologically normal sperm 93.85±0.08, 90.70±0.11, 89.08±0.10 and 87.66±0.10; intact acrosome 94.40± 0.12, 88.22±0.13, 84.60±0.14 and 81.70±0.17, and HOS reactive sperm 79.35 ±0.24, 65.99±0.31, 57.69±0.36 and 50.70±0.43, respectively, all of which decreased gradually and highly significantly (P is less than 0.01) with increase in storage time. The results were significantly (Pis less than 0.05) superior with Optixcell followed by TFYG extender than with the Bioxcell. The overall mean percentages of sperms with abnormal head, mid-piece and tail and those with different forms of acrosomal defects like swollen, ruffled, detached and denuded acrosome also followed the pattern of morphologically normal sperm and intact acrosomes. The values of all these traits increased significantly (Pis less than 0.05) with increase in storage duration at 5°C. The sperm protective ability of Optixcell extender was superior followed by TFYG and the least of Bioxcell. The Optixcell, a recently launched improved soybean milk based extender, excelled the Bioxcell extender and was statistically similar to TFYG in respect of all the sperm quality parameters to satisfactorily preserve the quality of buffalo semen at refrigeration temperature up to 3 days.

Cryopreservation of white-tailed deer (Odocoileus virginianus) semen using soybean-, liposome-, and egg yolk-based extenders

The objectives of the present study were to compare the use of soybean-based (Andromed), liposome-based (Optixcell), and egg yolk-based (Ovine Red, Triladyl, and Biladyl) extenders for cryopreservation of white-tailed deer semen. In experiment 1, ejaculates obtained from six bucks were aliquoted into the following extenders: Andromed, Ovine Red, Triladyl, and Biladyl (containing 4%, 6%, or 8% of glycerol). In experiment 2, ejaculates obtained from eight bucks were divided amongst Andromed, Ovine Red, and Optixcell extenders. Total and progressive sperm motility were assessed for each sample before and after cryopreservation using a computer-automated semen analyzer. In experiment 2, flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). In experiment 1, both Andromed and Ovine Red extenders exhibited higher post-thaw total motility than Biladyl containing 4% or 6% of glycerol (p<0.05). Andromed also produced higher progressive motility than all other extenders (p<0.01) before and after cryopreservation with no differences amongst the other extenders (p≥0.11). In experiment 2, there were no differences in total and progressive motility between Andromed, Ovine Red, or Optixcell extenders (p≥0.39). Additionally, there were no differences in sperm viability (p=0.18), acrosome integrity in viable sperm (p≥0.10), or DNA fragmentation index (p=0.15). These results demonstrated that soybean (Andromed) and liposome-based (Optixcell) extenders are equally as effective at cryopreserving white-tailed semen as egg yolk-based Ovine Red extender, but are superior to egg yolk-based Biladyl or Triladyl extenders.

OPTIXcell improves the postthaw quality and fertility of buffalo bull sperm

The present study was conducted to compare the liposome-containing, animal protein–free, commercially available OPTIXcell extender with the Tris–citric–egg yolk extender for postthaw quality and fertility of buffalo semen. Semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with an artificial vagina (at 42 °C) for 3 weeks (replicates). Semen ejaculates from each buffalo bull were divided into two aliquots and diluted (at 37 °C having 50 × 106 spermatozoa/mL) in the OPTIXcell or Tris–citric–egg yolk (control) extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours, and filled in 0.5-mL straws. The semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. The straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, the semen straws were thawed at 37 °C for 30 seconds to assess postthaw quality. Percentages of sperm motility, plasma membrane integrity, viability, and acrosomal integrity were improved (P < 0.05) in the OPTIXcell extender compared to the Tris–citric–egg yolk extender. Values for DNA integrity (%) did not differ in the OPTIXcell and Tris–citric–egg yolk extenders. The overall conception rate in buffaloes was improved (P < 0.05) with semen cryopreserved in the OPTIXcell extender (59.5%) compared to semen cryopreserved in the Tris–citric–egg yolk extender (41.5%). It is concluded that the liposome-containing commercially available OPTIXcell extender is more efficient to conserve postthaw quality and resulted in higher fertility rate of buffalo in the field.

Relative efficacy of egg yolk and soya milk-based extenders for cryopreservation (-196°C) of buffalo semen.

Aim:The aim was to compare commercially available soybean milk-based extenders, viz. Bioxcell® and Optixcell® (IMV, France) with standard Tris-citrate-fructose-egg yolk-glycerol (TFYG) extender for cryopreservation of buffalo semen.Materials and Methods:Semen was collected twice a week in artificial vagina from six sexually mature, 4-6 years old, healthy breeding bulls of Surti buffalo breed. In all 48 qualifying ejaculates (8 per bull) having initial motility >70% were split into three equal aliquots and were diluted (at 34°C keeping 100×106 sperm ml−1) in TFYG, Bioxcell and Optixcell extenders. The French mini straws filled from each aliquot were gradually cooled to 4-5°C, equilibrated at 4°C for 4 h and frozen in liquid nitrogen 2 vapor using programmable biofreezer. Just before freezing (post-equilibration) and 24 h after frozen storage, the samples were evaluated for various sperm quality parameters using standard protocols. Frozen semen straws were thawed in a water bath at 37°C for 30 s. The post-thaw incubation survival (37°C for 1 h) was assessed through motility rating at 0, 30 and 60 min of incubation.Results:The mean percentages of prefreeze sperms in TFYG, Bioxcell and Optixcell extenders in terms of progressive motility (69.48±0.37, 68.02±0.49, 70.94±0.38), viability (79.21±0.39, 77.38±0.48, 81.58±0.38), total abnormalities (7.90±0.14, 8.60±0.16, 7.08±0.15), intact acrosome (89.54± 0.18, 88.58±0.22, 90.52±0.21) and hypoosmotic swelling (HOS) reactivity (67.96±0.32, 65.65±0.42, 70.23±0.37) varied significantly (p<0.05) between extenders. Similar pattern of significant (p<0.05) variations between these extenders for post-thaw sperm progressive motility (47.71±0.79, 44.38±0.85, 49.90±0.90), viability (57.19±0.79, 53.85±0.84, 59.67±0.91), total abnormalities (12.33±0.17, 12.75±0.21, 11.27±0.18), intact acrosome (76.83±0.23, 75.90± 0.27, 78.50±0.25) and HOS reactivity (45.02±0.84, 42.31±0.82, 47.81±0.90) was also observed for TFYG, Bioxcell and Optixcell extenders. The recently launched improved soybean milk-based extender Optixcell excelled the older Bioxcell extender and even standard TFYG in respect of some of the sperm quality parameters.Conclusion:The advantages of soy lecithin-based bovine semen extenders over egg yolk regarding sanitary issues are unquestionable but still egg yolk-based semen extenders are widely used because of the cost factor and good in vivo fertility results.