Rutin protects boar sperm from cryodamage via enhancing the antioxidative defense

This study was aimed to investigate whether and how Rutin protects boar sperm against cryoinjury during cryopreservation. Five concentrations of Rutin with 0.2, 0.4, 0.6, 1.0, and 2.0 mM were added to the freezing extender of boar sperm, respectively, and the effects on quality and function of boar sperm after freezing‐thawing were assessed. The results showed that the sperm motility, mitochondrial activity, plasma membrane integrity, and acrosomal integrity were significantly improved in 0.4 mM and 0.6 mM Rutin groups (p < .05). Compared with ganoderma lucidum polysaccharide (GLP) or Tanshinone IIA, Rutin exhibited higher rates of mitochondrial activity and acrosome integrity (p < .05). Mechanistically, the addition of Rutin at the concentration of 0.6, 0.8, and 1.0 mM significantly attenuated ROS accumulation and MDA production by improving antioxidant enzymatic activity, including SOD, CAT, and GSH‐Px (p < .05). Functionally, a higher penetration rate and the increased total efficiency of fertilization were observed in the 0.4, 0.6, and 1.0 mM Rutin groups than in the control group (p < .05). Moreover, the addition of Rutin (0.6 mM) significantly induced an increase in both the cleavage and blastocyst rates (p < .05). In summary, supplementation with Rutin in cryopreservation medium protects boar sperm against ROS attack by enhancing the antioxidative defense.     

The positive effects of seminal plasma during the freezing process on cryosurvival of sperm with poor freezability in the rhesus macaque (Macaca mulatta)

The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (-0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (-16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.     

Effect of caspase inhibitor Z-VAD-FMK on bovine sperm cryotolerance

The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.     

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

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

解冻稀释液中添加精浆对冻融猪精子品质的影响

【目的】 探究冷冻前添加热休克蛋白A8(heat shock protein A8, HSPA8)和解冻后添加不同浓度精浆(seminal plasma, SP)对冻融猪精子的影响。【方法】 采用手握法采集长白猪精液, 添加0.5 μg/mL HSPA8到猪精液冷冻保护剂中进行细管分装, 投入液氮中保存3周后进行解冻, 解冻后添加不同浓度精浆(0、10%、30%和50%), 对冻融后长白猪精子的运动能力、质膜完整性、顶体完整性、细胞凋亡、线粒体膜电位、鱼精蛋白缺乏及体外获能水平等进行评估。【结果】 与对照组相比(无HSPA8和精浆), 添加0.5 μg/mL HSPA8处理组(无精浆)的精子直线速度(VSL)、曲线速度(VCL)、平均路径速度(VAP)和前向性运动(STR)均显著提升(P<0.05), 精子直线性运动(LIN)和运动的摆动性(WOB)均无显著差异(P>0.05);精子质量参数中活力、质膜完整性和顶体完整性均显著升高(P<0.05), 细胞凋亡水平与线粒体膜电位均显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。之后在解冻液中添加不同浓度的精浆, 与添加0.5 μg/mL HSPA8处理组(无精浆)相比, 精浆添加量达到50%时, 精子VSL、VCL、VAP、LIN、STR和WOB均显著提升(P<0.05);精子活力、质膜完整性、顶体完整性和线粒体膜电位均显著提高(P<0.05), 细胞凋亡水平显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。【结论】 在冷冻基础液中添加0.5 μg/mL HSPA8和解冻稀释液中添加50%精浆联合使用可以有效改善冻融精子质量, 将会对猪精液的冷冻保存及商业化生产提供一定的参考。     

Retracted: Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation

Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing‐thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 ± 0.026 g/l, and the TP after cryopreservation was 1.86 ± 0.6 g/l. The activities of acid phosphatase (0.82 ± 0.042 U/l), lactate dehydrogenase (234.4 ± 19.4 U/l), arylsulfatase (143.1 ± 32.5 U/l) and β‐N‐acetylglucosaminidase (58.39 ± 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen‐thawed semen samples (7.43 ± 0.64, 3224.6 ± 167.2, 422.6 ± 21.3 and 90.2 ± 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing‐thawing.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Addition of Glutathione to an Extender for Frozen Equine Semen

The manipulation of equine semen during cryopreservation reduces sperm viability and fertility because of, among other factors, membrane lipid peroxidation that makes cells highly susceptible to free radicals and reactive oxygen species (ROS). The oxidative effect caused by the generation of ROS can be reduced by the addition of antioxidants to the seminal plasma or to the extenders used for freezing. The current study was performed to test the in vitro effect of exogenous glutathione added in five different concentrations (control, 2.5 mM, 5.0 mM, 7.5 mM, and 10 mM [treatments 1-5, respectively]) to the extender for 12 stallions. Analyzed parameters were sperm motility, viability, and acrosome and plasmatic membrane integrity. Total motility was higher in treatments 1 and 2 (P < .05); viability, progressive motility, and plasmatic membrane integrity were higher in treatment 2 (P < .001). As for acrosome membrane integrity, treatment 3 showed the best results (P < .05). The addition of 2.5 mM glutathione to the freezing extender preserves total motility and increases sperm viability, progressive motility, and plasmatic membrane integrity. Concentrations above 2.5 mM were deleterious to spermatozoa.     

Effect of Seminal Plasma on Post‐Thaw Quality and Functionality of Corriedale Ram Sperm Obtained by Electroejaculation and Artificial Vagina

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post‐thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

Supplemented stallion seminal plasma can improve impaired motility due to the dilution effect in chilled Asian elephant sperm

The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.     

Cryopreservation of canine spermatozoa using a skim milk-based extender and a short equilibration time

Although useful spermatozoa cryopreservation techniques have been established, long-term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)-based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p < .05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p < .05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen–thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM-based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used.     

Effect of Different Levels of Glycerol and Cholesterol‐Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa

This study was conducted to determine the optimum level of glycerol and cholesterol‐loaded cyclodextrin (CLC) in a Tris‐based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10⁶ cells in Tris‐based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin–nigrosin staining) and functional membrane integrity (hypo‐osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10⁶ sperm. Post‐thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3‐h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10⁶ cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.     

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low‐density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer‐assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo‐osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC‐Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP‐based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.     

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.     

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl^® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.     

Linseed oil in boar's diet improved in vivo fertility and antioxidant status

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0 h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography–mass spectrometry (GC–MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0 h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.