Seminal plasma removal for medium-term preservation of ram sperm at 5 °C.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

In vitro aging of stallion spermatozoa during prolonged storage at 5°C.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca2+ ]i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca2+ ]i decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca2+ ]i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.

Short-term preservation of canine sperm-binding ability and other metrics using the INRA-96 in comparison to Tris-egg yolk extender.

The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA-96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA-96 extender as an alternative for the short-term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh and chilled-rewarmed samples, as well as the sperm-binding ability using the perivitelline membrane of the chicken egg as an indicator of the fertilizing capacity of the preserved semen. A total of 18 ejaculates from 9 French bulldogs (two ejaculates per dog) were collected and divided into two aliquots that were diluted in Tris-egg yolk 20% (control) or INRA-96 to a final concentration of 100 × 106 sperm/mL. Samples were refrigerated in a biological incubator at 5°C and evaluated at 0, 24 and 48 h time points. Comparing the two treatments after 48 h of refrigeration, both extenders showed similar values (p < .5) for the majority of kinetic parameters, with the INRA-96 group promoting a total motility of 88.1 ± 2.9%. In addition, the morphology, integrity and functionality of the plasma membrane were preserved above 70% in this group. Dilution with INRA-96 also provided a significantly higher amount of sperm bound (256.2 ± 21.1) to the perivitelline membrane of the egg yolk compared to the sperm-binding rates (p < .05) achieved at the use of Tris-egg yolk (215.2 ± 21 bound spermatozoa) at 48 h. Our study proved similar functional properties of dog sperm cells treated with INRA-96 in comparison to commonly used home-made Tris-based extender during short-time storage.

Effects of water-soluble curcuminoid-rich extract in a solid dispersion form (CRE-SD) on the sperm characteristics, longevity and casein kinase II catalytic subunit alpha protein stability in chilled goat semen.

The present study evaluated the effects of water-soluble curcuminoid-rich extract in a solid dispersion form (CRE-SD) on goat sperm qualities and sperm protein CSNK2A2 expression during liquid storage. Semen was collected from five fertile goats, using an artificial vagina. Ejaculates with a motility above 70% were cooled to 4 °C using TRIS-citric acid-fructose diluent with 10% egg yolk containing various concentrations of CRE-SD (0, 0.1, 1, 10 and 100 µg/mL). Chilled sperm were evaluated for sperm characteristics, casein kinase II catalytic subunit alpha (CSNK2A2) protein level and oxidative status up to 15 days. After 12 days of preservation, sperm motility, viability, acrosomal integrity and mitochondrial activity were significantly higher in the group preserved with 10 µg/mL CRE-SD as compared with the control group. Supplementation of CRE-SD at this concentration was also able to conserve the CSNK2A2 a significantly higher than that in control group until 9 days of cold storage, possibly by reducing oxidative stress. The molecular mass of the sperm CSNK2A2 protein detected in this study was 37 kDa; it was mostly located in the post-acrosomal region, midpiece and flagellum. These results demonstrate the possibility to use the CRE-SD as a natural antioxidant during liquid semen storage in goats.

Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares.

The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two groups in the ratio of the foals born (P > 0.05). The comparison of medians for the number of insemination cycles did not show significant differences. However, a significant difference (Kruskal-Wallis test, P = 0.014) was found in the number of the inseminations per conception in favour of frozen semen (2.5 vs. 1.8 with fresh chilled and frozen semen, respectively). The Cox regression revealed that the type of semen has a significant impact (P < 0.001) on the service period (duration of the insemination period): the use of frozen semen prolonged the insemination period. This could be due to management issues, since re-insemination with frozen semen took place after only one/a few missed oestrous cycles not used for AI.

Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in chilled and cryopreserved bull sperm.

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental-Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris-egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen-thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris-egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.

Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender.

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.

Effects of a simple method for determining the time of insemination and different methods on artificial insemination in common bottlenose dolphins (Tursiops truncatus).

In this study, we examined the usefulness of a simpler and more feasible method for determining the optimal timing of artificial insemination and the conditions for its success in six female common bottlenose dolphins. Pregnancy was successfully achieved in five dolphins by performing intrauterine insemination, using chilled semen stored for less than 3 days or frozen semen within 24 hr of exhibiting a peak serum estradiol (E2) level of 100 pg/mL or higher or on the day with a serum E2 level of approximately 100 pg/mL, measured with a simple measuring device. We concluded that the determining the optimal timing of intrauterine insemination by measuring serum E2 levels is a simpler and more useful method compared with the conventional approach.

Mitochondria-targeted antioxidant "MitoQ" improves rooster's cooled sperm quality indicators and reproductive performance.

The cell membrane of rooster sperm is sensitive to cold due to the high content of polyunsaturated fatty acids, which are very susceptible to lipid peroxidation. The present study was conducted to determine the effect of different concentrations of the mitochondrial-targeting antioxidant "MitoQ" on sperm quality and fertility potential of chilled semen in roosters. Semen samples were collected from 10 roosters, diluted in Lake extender, assigned into 5 groups according to MitoQ concentrations (0, 1, 10, 100 and 1000 nM MitoQ) and stored at 5 °C up to 48 h. Motility, mitochondrial activity, viability, membrane integrity, and lipid peroxidation were assessed at 0, 24, and 48 h of cold storage periods. In addition, the fertility potential was assessed using 24 h-cooled semen samples. Our results showed that extender supplementation with MitoQ had no effect (P > 0.05) on chilled semen samples quality parameters at time 0, while at times 24 and 48 h storage, samples contained 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, viability and membrane integrity compared to the other groups. In addition, semen samples containing 10 and 100 nM MitoQ showed higher (P ≤ 0.05) mitochondrial activity and lower (P ≤ 0.05) lipid peroxidation than other groups at 24 and 48 h storage. Fertility rate was higher (P ≤ 0.05) when the hens were artificially inseminated with 24 h-chilled semen samples containing 100 nM MitoQ. In conclusion, supplementing Lake Extender with 100 nM MitoQ could be a helpful strategy to preserve chilled semen quality and fertility potential in the rooster.

Effect of Vitamin D3 on some Antioxidant Parameters in Chilled Semen in Awassi Ram.

To sustain the viability of the sperm of farm animals, the sperm is chilled. However, reactive oxygen species (ROS) may damage it, resulting in oxidative stress and decreased sperm viability. This study aimed to assess the various concentrations of vitamin D3 as antioxidants in the chilled sperm of Awassi. This study was performed on 23 ejaculates from three Awassi rams. The samples were combined, diluted with Tris-egg yolk extender (1:10), and then divided into aliquots. Aliquots were treated with three vitamin D3 concentrations (T1=0.02, T2=0.004, and T3=0.002 g/ml) and one control without the addition of vitamin D3. The experimental and control groups were chilled to reach 5 ºC. Following treatment, the samples were centrifuged at 2,000 RPM for 20 min at 0 and 72 h after the treatment. Until evaluation, the seminal plasm was stored in a freezer at 20 ºC. In this study, the antioxidant activity of vitamin D3 was evaluated using malondialdehyde (MDA), ROS, total antioxidant capacity (TAC), superoxide dismutase (SOD), and catalase (CAT). The SAS software was used to analyze variance on repeated measures with a single factor. The results indicated that the TAC and SOD were considerably higher in T1, compared to that in T0, T1, and T2. In addition, CAT was considerably higher in T2 than in T0, T1, and T3. However, ROS and MDA did not differ significantly among the experimental groups. Despite the absence of a statistically significant difference among experimental groups, MDA decreased quantitatively on T1, relative to other experimental groups. In conclusion, a deficiency in vitamin D3 has a potential antioxidant capability, introducing a novel method for extending sperm storage.

Bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage with or without antibiotics in a tropical environment.

BACKGROUND: In tropical environments, boar semen is prepared either from a boar on the same farm as the sow herd or collected in semen collection centers and then transported to other farms. Thus, the semen doses can be used for artificial insemination either immediately or preserved for 2-3 days. The present study investigated the bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage in semen extender with or without antibiotics in Thailand. M&M: In total, 20 Duroc ejaculates were collected. Each ejaculate was diluted in Beltsville Thawing Solution extender either with 0.25 g of gentamicin per liter (ANTIBIOTIC) or without gentamicin (NO-ANITIBIOTIC) to create semen doses containing 3,000 × 106 sperm/100 mL. These were stored at 17 °C for 4 days. Semen characteristics and total bacterial count (CFU per mL, log10) were measured after collection and during storage. RESULTS: Sperm viability was decreased by 6.4% for every 1.0 log10 increase in total bacterial count (p = 0.026) and Staphylococcus spp. were the most frequently isolated across ejaculates. Throughout the 4 days of storage, sperm motility, viability and acrosome integrity in the ANTIBIOTIC group were higher than those in the NO-ANTIBIOTIC group (p < 0.05), while the total bacterial count was lower (1.9 ± 0.1 versus 3.9 ± 0.1 log10, respectively; p < 0.001). Without antibiotic supplementation, the total numbers of bacteria counted on days 2 and 3 of storage were higher than those determined on days 0 and 1 (p < 0.001). Differences in semen quality were detected on days 2 and 3 between the NO-ANTIBIOTIC and ANTIBIOTIC groups in high-viability semen (p < 0.05). However, no differences in sperm quality between the NO-ANTIBIOTIC and ANTIBIOTIC groups were detected in the low-viability semen on each storage day (p > 0.05). On the last day of preservation, Globicatella sanguinis (57.2%), Delftia acidovorans (18.9%) and Micrococcus spp. (5.9%) remained as the top three most abundant contaminants in the semen with antibiotic. CONCLUSION: Our findings contribute new insights toward reducing antibiotics as well as rational antibiotic use in the boar AI industry. The growth of bacteria was significantly greater only after 2 days of preservation in the semen without antibiotic. For semen doses diluted from highly viable ejaculates, it is possible to store for 2 days without any antibiotic supplementation. Moreover, bacterial counts increased at the end of storage in the presence of gentamycin, suggesting the loss of bacteriostatic properties of gentamicin to the growth of bacteria during storage.

Effects of green tea polyphenols and α-tocopherol on the quality of chilled cat spermatozoa and sperm IZUMO1 protein expression during long-term preservation.

Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with α-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of α-tocopherol (0, 2.5, 5 and 7.5 µg/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 °C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using α-tocopherol at 5 µg/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 µg/ml α-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and α-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.

Fertility prediction in dairy goats from Murciano-Granadina breed: The role of sperm evaluation and female traits.

Fertility is one of the most economically important traits in farm animals, due to the direct and indirect costs associated to low pregnancy rates. Thus, one of the priority goals in animal reproduction is to predict the performance that the semen doses will have in vivo based on the quality values obtained in laboratory assays. Attempts have been made for getting a predictive model of fertility of frozen-thawed sperm in dairy goats, but similar studies have not been conducted for chilled goat buck sperm doses that are mostly used for artificial insemination in many countries including Spain. We study how parameters of in vitro sperm quality and characteristics of Murciano-Granadina dairy goats may affect the in vivo fertility obtained after artificial insemination with semen doses chilled at 4 °C. Moreover, this information was used for obtaining predictive models of the fertility. Sixty-three ejaculates from 13 males were used to prepare chilled doses for the insemination of 495 goats over 13 sessions. Fresh and chilled sperm were evaluated for motility and plasma membrane integrity with a computer-assisted sperm analysis system and flow cytometry, respectively. Fertility was determined at parturition, according to the kidding goats. Overall fertility was 59.6%. Pearson's correlation coefficients between in vivo fertility and quality variables of fresh sperm were not significant and were low (below 0.34 in absolute value) for chilled sperm. Females' characteristics had a low negative impact on fertility (correlation coefficients of -0.19 with age, -0.20 with parturitions and -0.11 with total milk yield obtained in the best lactation). Fixed and mixed logistic regression procedures were used trying to explain the fertility results. None of the models accurately predicted fertility, but the best models included the percentage of total motile sperm or average path velocity from fresh semen, age of the females and the session effect (uncontrolled environmental effects). These analyses showed that primiparous goats were 2.42 times more likely to get pregnant than goats that had kidded four or more times. Our field assay data on fertility in Murciano-Granadina dairy goats highlighted the importance of making quality controls of sperm, of choosing the doses presenting high percentages of motile sperm exhibiting regular trajectories and of selecting the youngest goats for AI, after their first kidding. Efforts should continue to obtain better predictive models for improving fertility in goat dairy herds.

Assessment of Chilling Injury in Boar Spermatozoa by Kinematic Patterns and Competitive Sperm-Oviduct Binding In Vitro.

Sensitive detection of chilling injury in boar spermatozoa is required to evaluate novel hypothermic preservation concepts. The study's aim was to examine whether analyses of motility patterns and sperm binding in a competitive oviduct explant assay (cOEA) sensitively detect chilling-induced alterations in sperm function. Semen samples (n = seven boars) were split into four subsamples by dilution either in Beltsville Thawing Solution (BTS) or Androstar® Plus and stored at 5 °C or 17 °C. Storage temperature had a significant effect on the distribution of spermatozoa in seven major kinematic clusters. The effect size of chilling at 5 °C as estimated by Cramer's V was higher (p < 0.05) in the BTS medium (0.21) compared to AndroStar® Plus (0.11). Spermatozoa extended in Androstar® Plus had higher relative binding capacity compared to sperm in BTS (p < 0.05). Binding indices correlated with the percentage of viable, acrosome-intact (r = 0.62) and motile spermatozoa (r = 0.72, both p < 0.001). The cluster size of sperm with slow, vigorous movement was negatively correlated with sperm-oviduct binding (r = −0.43, p < 0.05). In conclusion, the cluster analysis of sperm kinematics and competitive sperm oviduct binding in vitro present meaningful biological tests to assess novel concepts for hypothermic semen preservation.

A combination of taurine and caffeine maintains sperm quality in equine semen during chilled storage.

OBJECTIVE: The objective of this study was to evaluate the effects of caffeine and taurine on the motility and viability of chilled equine semen. MATERIALS AND METHODS: A total of 12 ejaculates were collected from three mature stallions with proven fertility during the breeding season. The gel-free spermatic fraction of each ejaculate was divided into two aliquots and diluted with a semen extender (either INRA 96® or BotuSemen Gold®). The aliquots were then split and assigned to one of the six treatment groups: control (no supplement), caffeine (2 and 4 mM), taurine (25 and 50 mM), and a combination of caffeine (2 mM) plus taurine (25 mM). Samples were stored at 4°C and analyzed at different time points (0, 24, 48, 72, and 96 h) to evaluate total (TMOT) and progressive (PMOT) motility and viability by computer-assisted sperm analysis. RESULTS: Regardless of the extender, PMOT and TMOT decreased over time. However, compared with the control, the treatment with 4 mM caffeine significantly mitigated the decrease in PMOT at 72 h. Additionally, semen treated with a combination of caffeine plus taurine maintained a significantly higher PMOT at 96 h, with improved viability at all time points. CONCLUSIONS: The combination of caffeine plus taurine helps maintain chilled equine semen viability and progressive motility up to 96 h independently of the extender used.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen.

The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.

Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10-14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10), and T3 (d11-d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility.

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk-based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one "bad cooler" and one "good cooler" stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.

Optimization of cryopreservation protocols for cooled-transported stallion semen.

Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion sperm.

Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm recovered from the caudae epididymides. Ejaculated samples were split, and one portion was centrifuged and re-suspended to reduce seminal plasma content. All samples were diluted to 50millionsperm/ml and divided into two portions, one of which was supplemented with 0.5mM d-penicillamine. After 48h, 96h, 144h and 192h storage, sperm motility was assessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining, and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillamine had no effect on motility of ejaculated sperm (P>0.05) but reduced total and progressive motility of epididymal sperm. Sperm chromatin integrity was not influenced by storage time, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercial semen extender was neither beneficial nor detrimental to the maintenance of quality in ejaculated semen stored at 5°C. The negative effect on motility of epididymal sperm may reflect differences in (membrane) physiology of spermatozoa that have not been exposed to seminal plasma.