The effect of astaxanthin supplementation on the post-thaw quality of dog semen

Astaxanthin is a member of the carotenoid family well known for its anti-cancer, anti-diabetic, anti-inflammatory and antioxidant nature. This study was designed to investigate the effects of astaxanthin supplementation of the extender (buffer 2) on post-thaw dog semen quality. Semen from four healthy dogs was collected by digital manipulation twice a week. The ejaculates were pooled, washed, divided into four equal aliquots, diluted with the extender supplemented with different concentrations of astaxanthin (0, 0.5, 1 and 2 µM) and cryopreserved. The results showed that 1 µM astaxanthin was the optimum concentration that led to significantly higher (p < .05) post-thaw motility, kinematic parameters and viability than the other groups. In comparison with the control group, sperm samples supplemented with 1 µM astaxanthin showed significantly higher (p < .05) sperm counts with intact membranes (55.7 ± 0.6% vs. 51.3 ± 0.9%), intact acrosome (58.4 ± 0.7% vs. 53.5 ± 0.6%), active mitochondria (54.9 ± 0.5% vs. 42.6 ± 0.6%) and normal chromatin (67.6 ± 0.9% vs. 61.7 ± 0.6%). Furthermore, astaxanthin-supplemented samples showed significantly lower expression levels (p < .05) of pro-apoptotic (BAX), oxidative induced DNA damage repair (OGG1), oxidative stress-related (ROMO1) genes and higher expression levels of anti-apoptotic (BCL2), and sperm acrosome-associated (SPACA3) genes compared to the control. Thus, supplementation of 1 µM astaxanthin in semen extender results in improved freeze-thaw sperm quality of the dog.     

The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

Background

Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics).

Results

Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05).

Conclusions

These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics.     

The Effect of Trehalose Supplementation of INRA-82 Extender on Quality and Fertility of Cooled and Frozen-Thawed Stallion Spermatozoa

Stallion semen cryopreservation is often associated with poor post-thaw sperm quality. Sugars act as nonpermeating cryoprotectants. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thaw process. Semen samples were collected from six Arabian stallions, divided into five different treatments in a final concentration of 100 × 10⁶ sperm/mL by using INRA-82 extender containing 0, 25, 50, 100, and 200 mM of trehalose then subjected to both cold storage and cryopreservation. Sperm motility, acrosome, plasmatic membrane, and DNA integrity were analyzed, and 57 mares were used to evaluate the field fertility of chilled and frozen-thawed semen. Results showed that the extender containing 100 mM trehalose only increased the functional acrosomal, plasma membrane, and DNA integrities. The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw total motility compared to the control group, and chilled semen achieved higher pregnancy rates compared to the frozen-thawed one. Pregnancy rate of mares inseminated with frozen-thawed semen (P < .05; 46.15% vs. 36.36%, respectively) was lower than those inseminated with chilled semen (76.47% vs. 68.75%, respectively) but higher than control. In conclusion, addition of 50 mM trehalose yielded the highest quality stallion semen after cooling and post-thawing in terms of motility, integrities of acrosome, membrane, and DNA as well as improved field fertility.     

Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased (P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control (P < .05). A tendency (P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained <1% live acrosome-damaged spermatozoa. In the final experiment, the effects of hyaluronan supplementation post-thaw was investigated using hyaluronan concentrations of 100, 200, and 400 μg/mL. Motility parameters studied over an 8-hour period at 37°C yielded no consistent differences. In conclusion, addition of hyaluronan at a concentration of 200 μg/mL before freezing increased spermatozoal post-thaw motility. High concentration of hyaluronan (1,000 μg/mL) appeared to be detrimental to post-thaw motility. Effects of hyaluronan on fertility are beyond the scope of this study and have yet to be determined.     

The Effect of 2-Hydroxypropyl-β-Cyclodextrin on Post-Thaw Parameters of Cryopreserved Jack and Stallion Semen

Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

Effect of Melatonin Implantation to Sperm Donor Rams on Post-thaw Viability and Acrosomal Integrity of Sperm Cells in the Breeding and Non-breeding Season

The influence of melatonin administration to sperm donors on the freezability of ram semen and enzyme leakage through sperm cells during different steps of the cryopreservation process were evaluated in the breeding and non-breeding season. Melatonin implantation to rams in the breeding season improved post-thaw sperm viability and intact acrosome rates without influencing the motility rate (p   < 0.05). Likewise, the post-thaw alkaline phosphatase release through sperm cells was significantly lower in the melatonin-treated group in comparison with untreated controls (p   < 0.05). In the non-breeding season, melatonin administration enhanced intact acrosome rates (p   < 0.05) and reduced aspartate aminotransferase activity (p   < 0.05) post-thaw in the offseason ejaculates. Melatonin implantation twice in the breeding and non-breeding season did not produce any further improvement in the post-thaw sperm parameters in the non-breeding season ejaculates. It was concluded that melatonin administration to sperm donors improved freezability of ram semen collected from these rams and reduced enzyme leakage through sperm cells during cryopreservation.     

Effects of crocin on frozen-thawed sperm apoptosis,protamine expression and membrane lipid oxidation in Yanbian yellow cattle

Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Background

Proximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.

Methods

Three ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).

Results

Semen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).

Conclusion

Higher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.     

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 m m and Glu 5 m m . In study II, semen was collected twice and divided into three aliquots; control, AA 50 μ m and AA 250 μ m . Initial sperm motility was significantly higher in sperm diluted with AA 50 μ m ; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 m m .     

Sperm of the giant grouper: cryopreservation,physiological and morphological analysis and application in hybridizations with red-spotted grouper

In order to develop excellent germplasm resources for giant grouper (Epinephelus lanceolatus), cryopreservation of giant grouper sperm was examined in the present study. Firstly, 13 kinds of sperm dilution (ELS1-3, EM1-2, TS-2, MPRS, ELRS0-6) were prepared with physiological salt, sucrose, glucose and fetal bovine serum. The physiological parameters of ELRS3 (ratio of fast motion, ratio of slow motion, time of fast motion, time of slow motion, lifespan and motility) and ELS3 (sperm ratio of slow motion, time of slow motion and motility) were significantly higher than those of the other dilutions (P < 0.05). Secondly, after adding 15% DMSO and 10% FBS to ELRS3 and ELS3, most physiological parameters of frozen sperm were also significantly higher than the other gradients (P < 0.05), and sperm motility was as high as 63.68 ± 4.16% to74.75 ± 12.71% (fresh sperm motility, 80.70 ± 1.37% to 80.71 ± 1.49%). Mixed with the above dilutions, a final volume of 105 ml semen was cryopreserved. Finally, the sperm of giant grouper cryopreserved with cryoprotectants (ELRS3 + 15% DMSO + 10% FBS) was used for electron-microscopic observation and crossbreeding with red-spotted groupers (Epinephelus akaara). The electron-microscopic observation revealed that part of the frozen-thawed sperm was cryodamaged, e.g., flagellum fracturing and mitochondria falling out, while the ultrastructure of sperm membrane, mitochondria and flagellum remained intact. Also, the fertilization and hatchability rates of giant grouper frozen sperm and red-spotted grouper eggs were as high as 94.56% and 75.56%, respectively. Thus, a technique for cryopreservation of giant grouper sperm was successfully developed and applied to crossbreeding with red-spotted grouper eggs.     

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.     

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.     

Use of Density Centrifugation for Delayed Cryopreservation of Stallion Sperm: Perform Sperm Selection Directly after Collection or after Storage?

Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two‐layer iodixanol as well as single‐layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1‐day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two‐ and single‐layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30–40%. When cryopreservation was carried out after 1‐day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post‐thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post‐thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation.     

Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates

Background

Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to investigate the possibility of using these tests predictively.

Methods

Aliquots from fortyone ejaculates, obtained as part of the normal semen collection schedule at the Swedish National Stud, were used for morphological analysis by light microscopy, whereas thirtyseven were used for chromatin analysis (SCSA) by flow cytometry. The outcome of inseminations using these ejaculates was made available later in the same year.

Results

Ranges for the different parameters were as follows; normal morphology, 27–79.5%; DNA-fragmentation index (DFI), 4.8–19.0%; standard deviation of DNA fragmentation index (SD_DFI) 41.5–98.9, and mean of DNA fragmentation index (mean_DFI), 267.7–319.5. There was considerable variation among stallions, which was statistically significant for all these parameters except for mean_DFI (P < 0.001, P < 0.01, P < 0.001 and P < 0.2 respectively). There was a negative relationship between normal morphology and DFI (P < 0.05), between normal morphology and SD_DFI (P < 0.001), and between normal morphology and mean_DFI (P < 0.05). For specific defects, there was a direct relationship between the incidence of pear-shaped sperm heads and DFI (P < 0.05), and also nuclear pouches and DFI (P < 0.001), indicating that either morphological analysis or chromatin analysis was able to identify abnormalities in spermiogenesis that could compromise DNA-integrity. A positive relationship was found between normal morphology and pregnancy rate following insemination (r = 0.789; P < 0.01) and a negative relationship existed between DFI and pregnancy rate (r = -0.63; P < 0.05). Sperm motility, assessed subjectively, was not related to conception rate.

Conclusion

Either or both of the parameters, sperm morphology and sperm chromatin integrity, seem to be useful in predicting the fertilising ability of stallion ejaculates, particularly in determining cases of sub-fertility.     

Is Resveratrol Effective in Protecting Stallion Cooled Semen?

The aim of this work was to evaluate the effect of resveratrol (RSV) during liquid storage of stallion sperm for 24 hours at either 10°C or 4°C. The antioxidant RSV was added to reduce the oxidative damage that occurs during cold storage. Aliquots of 2 mL of diluted semen were stored either at 4°C or 10°C under anaerobic conditions, in the absence (control group) or presence of RSV at different concentrations (10, 20, 40, and 80 μM). Sperm quality parameters were assessed at 0 hours and after 24 hours of storage. Resveratrol treatment did not affect sperm quality parameters at 0 hours. At 24-hour storage, a significant (P < .01) decrease of sperm quality was observed independently from RSV supplementation and storage temperature. A significant decrease of viable spermatozoa with high mitochondrial membrane potential (SYBR+/PI−/JC-1+) was evident at 24-hour storage in 40- and 80-μM RSV groups compared with control group. Moreover, a decline of total motility in 80-μM RSV group compared with the control group and a decrease of progressive motility and average path velocity in 80-μM RSV group compared with control and 20-μM RSV groups were observed. In conclusion, our findings demonstrate that RSV supplementation does not enhance sperm quality of stallion semen after 24 hours of storage. Moreover, 40- and 80-μM RSV concentrations could damage sperm functional status, probably acting as pro-oxidant. Finally, although 24-hour storage significantly affected most of the sperm quality parameters, no significant differences were found in groups maintained at 4°C or 10°C, suggesting that stallion semen could be equally preserved at these different temperatures.     

Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization

During cryopreservation, spermatozoa may suffer cold and cryo-induced injuries ―associated with alterations in cell defense systems― that are detrimental to their function and subsequent fertility. This study aimed to determine the efficacy of supplementing the semen freezing extender with the antioxidant reduced glutathione (GSH) in cattle. Semen was collected from four bulls and diluted in a freezing extender supplemented with or without GSH (0, 1, 5, and 10 mM) before the cooling step of the cryopreservation process. After thawing, the quality of the frozen-thawed semen was investigated for motility, viability, acrosomal and DNA integrity, and subsequent embryo development after in vitro fertilization of bovine oocytes. Additionally, semen from one of the bulls was used to analyze semen antioxidative potential, sperm penetration into oocytes, male pronucleus formation rate, and embryo DNA integrity. The sperm quality varied among bulls after GSH supplementation. One bull had decreased sperm total motility, and two bulls had decreased sperm DNA integrity. GSH supplementation had positive effects on embryo development for three bulls. Two of them showed both improved cleavage and blastocyst formation rates, while the other one only showed an improved cleavage rate. We observed positive effects on early male pronucleus formation and no negative effects on DNA integrity and cell number in blastocyst stage embryos. Although the effect varies depending on individual bulls and GSH concentration, GSH supplementation in semen may improve in vitro embryo production from frozen semen.     

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 10⁶ sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 10⁶ spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.