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.     

Effect of nicotinic acid on the plasma membrane function and polyunsaturated fatty acids composition during cryopreservation in boar sperm

This study was conducted to evaluate the effect of nicotinic acid on plasma membrane integrity and fatty acid composition in frozen–thawed boar sperm. Boar semen was cryopreserved using freezing extender containing nicotinic acid (NA), then plasma membrane integrity, osmotic equilibration, lipid peroxidation and fatty acid were analysed. The plasma membrane integrity of frozen–thawed sperm was significantly higher in the 10 mM NA than in the 0 and 20 mM NA treatment groups (p < 0.05). Additionally, the osmotic equilibration ability was not different in treatment groups, but lipid peroxidation was significantly decreased in the 10 mM NA treatment group (p < 0.05). The saturated fatty acids were significantly decreased in the 10 mM NA treatment group, and C18:1n‐9, C18:2n‐6, C20:4n‐6, C22:5n‐6 and C22:6n‐3, and total polyunsaturated fatty acids (PUFAs) were significantly increased in the 10 mM NA treatment groups (p < 0.05). In summary, 10 mM NA improved plasma membrane integrity, inhibited lipid peroxidation and increased PUFAs in frozen–thawed boar sperm. These results suggest that NA may be useful to protect the plasma membrane and inhibit the loss of PUFAs for sperm cryopreservation in pigs.     

Resveratrol supplementation into extender protects against cryodamage in dog post-thaw sperm

Antioxidants have multiple protective roles in a variety of cells and thus can be used to protect sperm against cryo-damage during freezing, which affects fertility. The antioxidant resveratrol (3,5,4-trihydroxytrans-stilbene; RSV) has been reported to protect the animal sperm during cryopreservation, including human sperm. In this study, we assessed the protective effects of RSV supplementation on dog sperm cryopreservation. Semen was collected from four dogs and the effect of different concentrations of RSV (0, 100, 200, and 400 µM) on post-thaw sperm quality was examined. After thawing, sperm motility was assessed using computer-aided sperm analysis, and the structural integrity of the plasma membrane, acrosome, and chromatin was examined. In addition, their mitochondrial activity and gene expression were also assessed. Dog sperm cryopreserved with 200 µM RSV showed significant improvement in post-thaw sperm motility and viability compared with that of the control group (P<0.05). Moreover, RSV-supplemented samples showed significantly higher numbers of sperm with an intact plasma membrane, active mitochondria, and structural integrity of acrosomes and chromatin than that of control samples (P<0.05). Furthermore, gene expression showed that RSV supplemented samples showed lower expression of pro-apoptotic (BAX), reactive oxygen species (ROS) modulator oxidative stress-related (ROMO1) and 8-oxoguanine DNA glycosylase 1 (OGG1) whereas higher expression levels of anti‐apoptotic (BCL2), protamine-2 (PRM2), protamine-3 (PRM3) and sperm acrosome‐associated 3 (SPACA3) genes than control. Our results suggest that RSV, at its optimum concentration, can be efficiently used as an antioxidant in the cryopreservation of dog sperm.     

Evaluation of pentoxifylline and Basal Medium Eagle supplemented to diluent on cryopreserved goat spermatozoa

This study investigated the effect of pentoxifylline (PTX) and Basal Medium Eagle (BME) on frozen–thawed goat spermatozoa. Immediately after initial examination of ejaculated semen, samples were pooled and reexamined for quality. Then, samples were divided into eight equal aliquots and diluted with a basic tris-extender containing PTX (3, 6, 9 mM) and BME (5 mM) to reach a final concentration of 25 × 10⁹ and frozen. After 24 hr, the samples were individually thawed at 37°C for 30 s and evaluated for different characteristics. Obtained post-thaw results from Computer-Assisted Sperm Analysis indicate using of 3 and 6 mM PTX led significantly to an improvement in total motility, progressive motility and velocity characteristics of spermatozoa, except the beat/cross frequency (BCF) which indicated statistically no differences (p > .05) among control and treatments. Diluents prepared with BME (5 mM) and PTX alone (3 and 6 mM) improved significantly the membrane integrity–functionality, acrosome integrity and also hyaluronidase activity. Regarding recovery rate, the results showed significantly (p < .05) higher values for diluents containing 3 and 6 mM PTX compared to other groups. Malondialdehyde concentration exhibited also a significant difference (p < .05) in diluents supplemented with 5 mM BME, 3, 6 and 9 mM PTX, and mixture of 3 mM PTX and 5 mM BME which illustrate a similarity for active mitochondria, apoptotic-like and dead spermatozoa. Finally, the ratio of sperm chromatin dispersion stained spermatozoa presented significant differences (p < .05) among treatments in which the diluents added PTX alone demonstrated significantly lower values than control and extenders containing the mixtures of BME and PTX. In conclusion, the observation in this study indicates using of 3 and 6 mM PTX and BME alone may improve significantly (p < .05) the quality of cryopreserved goat spermatozoa.     

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.     

Antioxidative effect of BHA in soya bean lecithin‐based extender containing Glycerol or DMSO on freezing capacity of goat semen

The aim of this study was to evaluate the effects of butylated hydroxyanisole (0 or 4 mM) along with different concentrations (5 or 7%) of glycerol (G) and dimethyl sulphoxide (DMSO) as cryoprotectant (CPAs) on freezability of goat semen. Semen was collected from four bucks (3–4 years) twice a week for five weeks. The pooled ejaculates were diluted with extender containing two different concentrations of G or DMSO in combination with BHA. Afterwards, the diluted samples were loaded into 0.25 ml straws and frozen using a standard protocol. After thawing motility parameters, viability, membrane integrity and total abnormality were assessed. The Results showed that the presence of BHA in extender, type and level of CPAs as main factors had significant effects on goat sperm viability, total and progressive motility after freezing–thawing processes (p < .05). Also, the interaction of BHA (0 and 4 mM) and levels of G or DMSO (5 or 7%) had a significant effects (p < .05) on total motility, viability and some characteristic. In this case, the addition of 5% G or DMSO with BHA resulted in highest motility and viability than the other groups (p < .05). The addition of G5 (with and without BHA) increased VSL and reduced abnormality than the other groups (p < .05). The results showed that the main effects of CPAs and CPAs level on membrane functionality were significant (p < .05). Also there were no significance differences in the interactive effects of MDA, VCL, VAP, ALH, LIN and STR among the groups (p > .05). Finally, it can be concluded that the use of 5% CPAs with or without BHA may result in better post‐thaw sperm quality of goat.     

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.     

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p < .05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p < .05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p < .05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.     

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.     

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.     

Sperm subpopulations influence the pregnancy rates in cattle

This study aimed to study the characteristics and subpopulations of spermatozoa from bulls with low and high reproductive performance based on pregnancy rates. Based on historical records of pregnancy rate from four farms, 24 bulls were selected. Two groups were established, with low pregnancy rates (n = 12; LOW), including bulls that presented pregnancy rates <52.27% (33.33% to 51.81%); and a group with high pregnancy rates (n = 12; HIGH), with pregnancy rates >52.27% (52.27% to 69.64%), after fixed-time artificial insemination (FTAI). The thawed sperm straws were analysed to sperm kinetics, morphology, plasma membrane integrity and sperm subpopulations. The LOW group exhibited a higher proportion of static cells (p < .05). In contrast, the HIGH group showed greater percentages for membrane integrity and total and progressive motility, and cells with fast and medium velocity (p < .05). In the cluster procedures, four sperm subpopulations were established. The low-fertility bulls presented the highest percentage of subpopulation 2 (41.46%), characterized by slow and progressive spermatozoa. The high-fertility bulls exhibited the highest percentage of subpopulation 3 (37.17%), characterized by fast and nonlinear spermatozoa. Results from this study indicated that bulls with greater percentages of fast and nonlinear spermatozoa seem to have greater fertilization capacity and the subpopulations analysis can be considered a tool to identify ejaculates with high fertility.     

Orally administered Chrysin improves post‐thawed sperm quality and fertility of rooster

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post‐thawed sperm quality and fertility of Chrysin‐fed roosters were assessed in this study. Twenty 40‐week‐old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch‐0), 25 (Ch‐25), 50 (Ch‐50) or 75 (Ch‐75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post‐thawed samples) and mitochondrial activity (only in post‐thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post‐thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post‐thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post‐thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post‐thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post‐thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch‐50 and Ch‐75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post‐thawed spermatozoa was significantly improved in all Chrysin‐fed groups compared to Ch‐0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation‐induced impairment of sperm quality and fertility rate.     

Cryopreservation of llama semen using a combination of permeable cryoprotectants

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal–Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.     

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post‐thaw sperm quality of Holstein bulls

This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.     

The relationship between acrosome reaction and polyunsaturated fatty acid composition in boar sperm

This study investigated the relationship between acrosome reactions and fatty acid composition with respect to fertility in boar sperm. The acrosome reaction was induced more than 85% by 60 mM methyl-beta-cyclodextrin (MBCD), and plasma membrane integrity was significantly reduced dependent on the MBCD level in boar sperm (p < .05). The acrosome-reacted sperm exhibited significantly higher saturated fatty acids (SFAs) and lower polyunsaturated fatty acids (PUFAs) composition compared to the non-acrosome reaction group (p < .0001). In addition, the PUFAs, C22:5n-6 (docosapentaenoic acid [DPA]; p < .01) and C22:6n-3 (docosahexaenoic acid [DHA]; p < .0001) were significantly decreased, and cleavage and blastocyst formation of oocytes were significantly (p < .0001) decreased in acrosome-reacted sperm relative to non-acrosome-reacted sperm. Moreover, acrosome reaction was positively correlated with SFAs, whereas negatively correlated with PUFAs, of the PUFAs, the DPA (p = .0005) and DHA (p = <.0001) were negatively correlated with the acrosome reaction. Therefore, these results suggest that the PUFAs composition of sperm is closely involved in acrosome reaction in pigs.     

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.     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.     

Cell cycle synchronization and analysis of apoptosis‐related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna)

The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts.     

Supplementing a skimmed milk–egg yolk-based extender with L-carnitine helps maintain the motility,membrane integrity and fertilizing capacity of chilled ram sperm

This study examines the effect of L-carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA-FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5-15°C-2h) and at 5°C for 24 hr (LC5-5°C-24h) was tested in three ewe groups via cervical fixed-time artificial insemination. In two groups, the fertilizing capacity of the LC5-5°C-24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5-15°C-2h and LC5-5°C-24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk–egg yolk-based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.     

Influence of seminal plasma during different stages of bovine sperm cryopreservation

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 10⁶ sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (−65.2%), acrosomal membrane (−34.0%) and mitochondrial potential (−48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.