Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

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.     

荷斯坦公牛PRNP多态性与精子活力相关性及抗病性评估

为分析荷斯坦公牛朊蛋白基因(PRNP)多态性与精子活力的关系,并对其抗病性进行评估,选育抗病公牛,本实验以公牛精液为样品,研究脚基因中12bp、23 bp和24 bp 3个片段的插入/缺失多样性、基因mRNA转录水平及其与精子活力等指标的关系.结果表明,12bp和23 bp在群体中均得到3种基因型,24 bp只有2种基因型.不同基因型群体的mRNA转录水平存在显著差异,而基因型与精液产量和活力等指标存在相关性,抗病力和精子活力指标存在负相关.本研究中3个片段的插入-缺失多样性可以作为公牛选育的辅助标记.     

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential,membrane integrity,sperm motility and in vitro fertilization in bovine epididymal sperm

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post‐mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty‐six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer‐assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre‐freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post‐thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.     

三种因素对雄蜂精子数量和活动率影响的研究

本文较详细地研究了雄蜂体重、性成熟程度和天气情况对雄蜂精子总数和精子活动率的影响,结果表明:雄蜂总精子数与雄蜂的体重呈正相关。性成熟很充分的雄蜂其精子总数相对较多,但精子活动率却低于性成熟一般的雄蜂。虽然在不同的天气里雄蜂精子总数差异不大,但精子活动率有着比较明显的差异。本实验旨在采集到高质量的雄蜂精液,为蜜蜂人工授精技术的发展提供理论依据。     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.     

Validation of a commercial human ELISA to measure hyaluronic acid concentration in feline plasma

Our goal was to validate a human hyaluronic acid (HA) ELISA (Hyaluronic acid plus ELISA; TECOmedical Group) for use in feline plasma. Plasma from 5 healthy cats and 5 critically ill cats was used for validation of the assay. Validation methods performed included intra- and inter-assay variability, spike-and-recovery, and dilutional linearity. All measurements were performed in duplicate. The precision study revealed good intra-assay CV of 7.4–8.9%; inter-assay CV was 3.4–4.2%. Extraction efficiency via spiking tests yielded mean recovery of 89.6%. The assay met criteria for acceptable linearity using 3 serial dilutions. Our results demonstrate that this commercial HA ELISA had acceptable analytical performance using feline plasma and could be a useful tool in the veterinary clinical research setting.     

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10^–3, 1 × 10^–4, 1 × 10^–5, 1 × 10^–6 M) or Lyc (0, 1 × 10^–4, 1 × 10^–5, 1 × 10^–6, 1 × 10^–7). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10^–3 M VC or 1 × 10^–4 M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10^–4 M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10^–3 M VC or 1 × 10^–4 M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.     

纤维蛋白粘合剂对犬皮肤切口透明质酸和羟脯氨酸含量的影响

为探讨自制纤维蛋白粘合剂的作用机理,将其应用于犬皮肤切口,并以常规手术缝合切口为对照进行体内粘接效果对比试验。选取健康犬20只,随机分为缝线组与粘合剂组,分别采用丝线和自制纤维蛋白粘合剂闭合皮肤切口,测定皮肤切口中的透明质酸（HA）和羟脯氨酸（HYP）含量。结果表明,愈合过程中,粘合剂组HA含量在术后2d呈一过性升高,HYP含量在术后2d后持续升高,8d含量达到最高值,与缝线组比较差异显著（P〈0．05）。     

Evaluation of the inflammatory response to two intra-articular hyaluronic acid formulations in normal equine joints

Intra-articular (IA) hyaluronic acid (HA) is commonly used to treat equine arthritis. Inflammatory response or “joint flare” is a recognized potential side effect. However, the incidence and severity of inflammation following IA HA injection in horses is not well documented. This study compared the effects of two IA HA formulations of different molecular weight (MW) and a saline control on clinical signs and synovial fluid markers of inflammation in normal equine joints. Eight adult horses each had three healthy fetlock joints randomly assigned to treatment with either 1.4 mega Dalton HA, 0.8 mega Dalton HA or saline control once weekly for three weeks. Clinical evaluation and synovial fluid analysis were performed by blinded assessors. Outcomes of interest were lameness score, joint effusion score and synovial fluid white cell count and differential, total protein, viscosity and serum amyloid A. Joints injected with HA developed significant mild-to-moderate inflammatory responses often associated with lameness and joint effusion compared with saline control joints. The higher MW HA formulation elicited a significantly greater inflammatory response than the lower MW HA after the first injection. In HA injected joints, viscosity remained poor for the entire study. Both IA HA formulations in this study induced an inflammatory response in healthy equine joints. This may have implications for the use of HA in equine joints. The findings in this study are limited to the two HA formulations used. Further investigation of different HA formulations and the use of HA in normal and arthritic equine joints is warranted.     

Follicular fluid stimulates capacitation and acrosome reaction in alpaca sperm (Vicugna pacos)

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (<3 mm), medium (3‐6 mm) and large (>6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa.     

Effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters

The objective of this study was to evaluate the effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters. Twelve Ross 308 broiler breeder roosters (age: 52 weeks; weight: 4,900 ± 210 g) haphazardly allotted to three dietary treatments (each treatment contained four replicates and one bird in each replicate) for six weeks. Treatments were different levels of flaxseed (0% flaxseed [GFL0], 2% flaxseed [GFL2] and 4% flaxseed [GFL4]). The feed intake quadratically decreased (p < .05) with increasing whole flaxseed levels for the period (58 to 60 weeks). Sperm traits (semen volume and sperm concentration, sperm total and forward motility, sperm viability and morphology, sperm plasma membrane functionality) were evaluated every two weeks (four times), and sperm fatty acid profile was assessed at the end of the experiment. Semen volume, sperm concentration and sperm morphology were not affected by treatments. On week 60, GFL2 group showed a significantly lower percentage of total and progressive sperm motility and sperm membrane functionality in comparison with the control and GFL4 groups. Also, sperm viability was lower in GFL2 group compared with other groups on week 58 (p < .05). In terms of sperm fatty acid profile, GFL2 group significantly reduced the percentage of linoleic acid (C18:2 [n-6]) in comparison with other groups. However, any of the other fatty acids were not affected by dietary flaxseed. In conclusion, dietary supplementation of whole flaxseed could not improve the quality of aged broiler breeder roosters' sperm in this study, nor it could alter the sperm fatty acid profile; thus, it seems necessary to use some antioxidants such as vitamin E in the diet of aged broiler breeder roosters, when supplementing the diets with oils or oilseeds such as flaxseed.     

Comparing accuracy of fish sperm motility measurements obtained from two computational extremes in tracking approaches: Nearest neighbor and multiple hypothesis tracking

We conducted a study to assess the accuracy of nearest neighbour (NN) and multiple hypothesis tracking (MHT) methods—which are opposite extremes in computational complexity—in determining the percentage of motile sperms and the number of sperms tracked in simulated data of fish sperm movements, and to evaluate the resulting number of tracking errors and analysis duration. Sperm tracking and swimming path assembly were assessed in 36 video clips (1 s length at 100 fps) of emulated Rhamdia quelen sperm kinetics at different densities (50, 100, 200 or 300 spermatozoa in the field of view) and motility rates (30, 60 or 90%). The MHT method accurately estimated the percentage of motile sperms, whereas NN underestimated it by up to 6.59%. Increase in sperm density reduced the number of sperms tracked from both trackers. With more than 50 sperms in the field of view, NN and MHT tracked 73% and 92% of the ground-truth sperm count, respectively. Both trackers showed a quadratic increase in tracking errors with increasing sperm density. The maximum percentage of errors at 90% motility was 12% for NN and 4.7% for MHT. The MHT tracker required up to 150 s to track 300 sperms, whereas NN completed the tracking procedure in less than 0.5 s. On maintaining a density of up to 100 sperms in the field of view, it was possible to obtain high accuracy, low number of tracking errors and an acceptable analysis duration with both tracking methods.     

Combined effect of apigenin and ferulic acid on frozen‐thawed boar sperm quality

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high‐efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen‐thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen‐thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen‐thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen‐thawed boar sperm.     

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.     

Effects of exposure to methylglyoxal on sperm motility and embryonic development after fertilization in mice

Methylglyoxal (MG) is a precursor for the generation of endogenous advanced glycation end-products involved in various diseases, including infertility. The present study evaluated the motility and developmental competence after in vitro fertilization of mouse sperm which were exposed to MG in the capacitation medium for 1.5 h. Sperm motility was analyzed using an SQA-V automated sperm quality analyzer. Intracellular reactive oxygen species (ROS), membrane integrity, mitochondrial membrane potential, and DNA damage were assessed using flow cytometry. The matured oocytes were inseminated with MG-exposed sperm, and subsequently, the fertilization and embryonic development in vitro were evaluated in vitro. The exposure of sperm to MG did not considerably affect the swim-up of sperm but resulted in a deteriorated sperm motility in a concentration-dependent manner, which was associated with a decreased mitochondrial activity. However, these effects was not accompanied by obvious ROS accumulation or DNA damage. Furthermore, MG diminished the fertilization rate and developmental competence, even after normal fertilization. Collectively, a short-term exposure to MG during sperm capacitation had a critical impact on sperm motility and subsequent embryonic development after fertilization. Considering that sperm would remain in vivo for up to 3 days until fertilization, our findings suggest that sperm can be affected by MG in the female reproductive organs, which may be associated with infertility.