The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols within each extender. Mean TM was greater in samples diluted with STAR than in samples diluted with Botucrio (P ˂ 0.05), but no significant differences were observed for this variable between the other studied extenders. From all evaluated samples, twenty ejaculates showed the greatest TM when using the lyophilized extenders and the CP1. Thus, lyophilized extenders are a promising option for stallion sperm cryopreservation and have the advantage of storage and distribution at room temperature for at least one year.

This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane integrity, lipid peroxidation, and DNA fragmentation. The findings indicated that 25-100 μg/mL of silymarin significantly improved viability and mitochondrial membrane potential while reducing the stallion sperm lipid peroxidation, DNA fragmentation, and apoptosis compared with the control group (p < 0.05). Silymarin concentrations of 75 μg/mL and 100 μg/mL significantly increased progressive motility and plasma membrane integrity (p < 0.05). Based on our findings, it can be inferred that silymarin exhibited a dose-dependent enhancement in the frozen-thawed Arabian stallion sperm quality. The most favorable outcomes were observed when 100 μg/mL silymarin was used.

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation on sperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM of stored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation (SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 °C before SC; iv) Storage for 8 h/5 °C before SLC; v) Storage for 8 h/15 °C before SC; and vi) Storage for 8 h/15 °C before SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classified as \"good freezers\" (GF) or \"bad freezers\" (BF). The PM in samples immediately frozen was greater than in the stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage, respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂ 0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation. Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storage conditions (p ˂ 0.05). All BF stallions \'showed post-thaw PM ˂ 30 % when samples were previously stored. Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selection technique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage, although BF semen should be prepared by SLC immediately after collection.

Semen cryopreservation is an important technique for preserving the genetic material of numerous species. However, frozen semen is highly susceptible to sperm DNA damage and reduced motility, resulting in decreased fertility. The standard method for cryopreservation and several approaches have not been elucidated. This study aimed to determine the effects of supplementing rooster semen extender with a combination of phosphorus and vitamin B12 on cryopreserved semen quality. Semen was collected weekly via dorso-abdominal massage from 57 Burmese × Vietnam-crossbred Thai native roosters aged 1-3 years. In total, 139 semen samples were collected, pooled, and diluted to 200 million sperm per dose. The pooled sample was divided into six experimental groups: a control group (0.00%) diluted with modified Beltville Poultry Semen Extender (BPSE) and five treatment groups diluted with modified BPSE supplemented with phosphorus and vitamin B12 at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%, respectively. The semen samples were frozen and evaluated at 0, 15, and 30 min after thawing. Sperm kinematic parameters were determined using a computer-assisted sperm analysis system. Sperm quality was evaluated by measuring sperm viability, mitochondrial activity, acrosome integrity, and plasma membrane integrity. Statistical analyses were performed using a general linear mixed model (MIXED) in SAS. Factors in the statistical model were experimental groups, time after thawing, and interaction between experimental groups and time after thawing. Total and progressive motilities were greater in semen supplemented with 0.04% phosphorus and vitamin B12 compared with those in the control (p < 0.05). At 15 min post-thawing, VCL, VAP, and HPA in the 0.04% phosphorus and vitamin B12 supplementation group was greater than that in the control (p < 0.05). Phosphorus and vitamin B12 supplementation did not affect sperm kinematics at 0 and 30 min after thawing (p > 0.05). All the sperm parameters that were tested for the 0.04% phosphorus and vitamin B12 supplementation group in modified BPSE were the highest at all the timepoints after thawing. Thus, supplementing frozen semen extender with 0.04% phosphorus and vitamin B12 increased sperm motility, sperm kinematic parameters, and sperm quality.

BACKGROUND: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C.
OBJECTIVE: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C.
METHODS: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode\'s (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode\'s 67 mm glucose-10 mm pyruvate and modified Tyrode\'s 67 mm glucose, and metabolic analysis conducted.
RESULTS: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose-10 mm pyruvate/10 μm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+, pyruvate, lactate, and ATP.
CONCLUSIONS: Aliquots stored in a 67 mm glucose-10 mm pyruvate-based medium supplemented with 10 μM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+.

This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese × Vietnam crossbreed) were included. Semen was collected by applying dorsal-abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into 6 groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2 to 6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%. Semen fertility ability was tested in 6 replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to 3 groups (control, 0.04, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59 vs. 48.50%: P < 0.05) and hatching rates (58.80 vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.

The identification of different morphometric patterns of spermatozoa serves as a basis for improving our understanding of the diversity in an ejaculate and to relate them to the potential fertility of males. In this study, we aimed to examine the semen subpopulation structure, following dilution in semen of extenders, using a mathematical approach a possible application to fertility analyses. Ten sexually mature Bos taurus bulls were randomly allotted to one of three groups: (1) Tris-citric acid-egg yolk extender (Tris-EY); (2) commercial egg yolk extender OptiXcell® and (3) commercial egg yolk extender Triladyl®. The results showed significant differences (p < .05) between extenders in terms of values for head size and head shape variables of individual sperm, indicating an influence of extender composition. Sperm head width was found to significantly differ (p < .05) according to the extender, decreasing in the following order: OptiXcell® (4.836 ± 0.017 μm), Triladyl® (4.695 ± 0.012 μm) and Tris-EY (4.638 ± 0.010 μm). Principal component analysis allowed us to identify two subpopulations in OptiXcell®, and three subpopulations were each found in Triladyl® and Tris-EY. Overall, we observed significant differences between sperm subpopulations within each extender (p < .05), with differences in sperm head size and shape between bovine species that can be related to functionality and fertility capabilities.

Equilibration with an extender is necessary to allow cryopreservation of bovine sperm. The aim of trial 1 was to assess the effect of 24 h versus 4 h equilibration time with three different extenders on sperm quality and to select the preferred extender for each bull. The aim of trial 2 was to investigate the effect of using a 24 h equilibration time with a bull-specific extender on field fertility. For trial 1, three ejaculates each from eight Holstein Friesian breeding bulls were used as the split-sample, including two equilibration times (4 h and 24 h) and three extenders (BioXcell, Triladyl, and OptiXcell). For trial 2, from 5 to 10 ejaculates from the same bulls were collected and treated (split-sample) as BioXcell with 4 h equilibration and either Triladyl or OptiXcell, both with 24 h equilibration. A total of 11,059 straws were used for insemination of cows and heifers. For Triladyl, progressive sperm motility, acrosome defects, and plasma membrane and acrosome integrity improved with a 24 h compared to a 4 h equilibration time. Four bulls each were used with Triladyl and OptiXcell for trial 2. In trial 2, non-return rates did not differ among groups. Therefore, using a 24 h equilibration time might improve in vitro sperm parameters, depending on the extender used. Moreover, it would be possible to change from 4 h to 24 h equilibration time without impairing field fertility.

This study evaluated the effect of sperm concentration of boar semen doses on their capacity to maintain its motility over the thermo-resistance test (TRT; sperm resilience) and verified if the extender type (short or long-term) could influence this effect. Thirty ejaculates from five crossbred mature PIC® boars were used, and a factorial design was followed to produce semen doses with 1.5 billion cells in 45 or 90 mL, using Beltsville Thawing Solution (BTS) or Androstar® Plus (APlus). Then, low-concentration doses (16.7 × 106 cells/mL in 90 mL) and higher-concentration doses (33.3 × 106 cells/mL in 45 mL) with BTS or APlus were produced and stored at 17 °C for 168 h. At 72 h, during the TRT, the low-concentration doses (16.7 × 106 cells/mL) lost three-fold less motility than doses with 33.3 × 106 cells/mL (P < 0.01), regardless of the extender type (11. 5% vs. 30.5% of initial motility, respectively). Similar results were found when the TRT was carried out at 168 h, with low-concentration doses losing two-fold less motility (11.4%) than highly concentrated doses (25.9%; P < 0.01). No sperm concentration effect was observed on membrane integrity or mitochondrial membrane potential (P ≥ 0.23). The osmolarity was not affected by the sperm concentration (P = 0.56), only by the extender and the storage time (P < 0.01). In conclusion, the sperm concentration effect on sperm quality was not influenced by extender type, and the data suggest that a low concentration of semen doses positively affects sperm resilience.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106  sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.