Pig Reproductive and Respiratory Syndrome Virus (PRRSV) Does Not Attach to Boar Sperm;It Affects Only the Velocity Pattern and the Mobility Pattern

The purpose of the study was to evaluate the sperm viability of semen infected with PRRSV viral particles, observing the effect of the Virus on the motility of boar sperm. The work was carried out at the FMVZ-BUAP Genetics and Reproduction Laboratory. 5 stallions were used. Each sample contained 1 × 106 sperm, the PRRS virus strain was ATCC-VR-2332 (0, 102, 104 and 106 copies of RNA/mL in triplicate), it was observed daily at the CASA;Hamilton Thorne®. Cells with MT (P < 0.05) on days 1, 3, 5, 7 and 10 of evaluation with 201 ± 7.3, 167 ± 10.1, 165 ± 14.6, 134 ± 8.2 and 120 ± 8.8, respectively. The % MP between control and virus concentrations (P ≥ 0.05). The LCV on day 1 and 7 PI at 10X2 and 10X6 (P < 0.05) vs control. In the Correlation Matrix, where it is observed that there is a correlation between VSL and VAP, VSL and VCL, VCL and ALH, VAP with ALH. There is a correlation of VSL and ALH, STR and ALH. In this study there were (P ≤ 0.01) in the VCL, in the concentrations (102) 162.81 ± 10.65 and (106) 177.12 ± 5.77 vs 193.04 ± 4.62 of control. This indicates that altering these parameters would be related to fertility and the PRRS virus affects the LCV. Regarding the VSL, it was observed that the sperm infected with viruses 102, 104 and 106 of 48.00 ± 3.38, 49.88 ± 1.83 and 50.55 ± 2.24 Vs. 56.66 ± 1.68 of control respectively, the control would have greater possibilities of fertilizing the oocyte. In this study, it was found (P ≤ 0.01) in the VAP with 102 of 77.26 ± 5.16, 104 with 83.35 ± 2.41 and 106 with 81.29 ± 3.14 vs the control with 90.56 ± 2.07. Regarding the ALH there is (P < 0.05) a 104 with 8.70 ± .26 and 106 with 9.64 ± 0.23 vs control 8.50 ± 0.27. The presence of different concentrations of PRRSV in boar semen induces changes in different types of sperm motility. Infection of ejaculates with the PRRS virus affects sperm motility on days 1, 3, 5, 7, and 10 post-infections.

Effect of Cryopreservation on DNA Integrity and Morphological Structure of Boar Sperm

The paper was to explore the effect of cryopreservation on DNA integrity and morphological structure of boar sperm, and to explore the protective effect of trehalose and lactose on frozen boar sperm. The morphology, ultrastructure and DNA integrity of sperms were observed by phase contract microscope, scanning electron microscope （SEM） and acridine orange （AO） staining, respectively. The results showed that the normal morphological rate and DNA integrity rate of fro- zen sperms were significantly lower than that of fresh sperms （95.5% and 94.7%, respectively）, and the difference between two frozen groups was also significant （P 〈 0.05 ）. The normal morphological rates in trehalose group and lactose group were 74.7% and 67.6%, while DNA integrity rates in trehalose group and lactose group were 66.4% and 63.2%, respectively. The common deformations of frozen sperms under SEM were partial or complete fracture between head and neck, swollen neck_ dama=ed aemsome stnJetn~.

Fluorometric Viability Assessment of Capacitated and Acrosome-Reacted Boar Spermatozoa by Flow Cytometry

Sperm capacitation involves functional changes, such as the removal or appearance of specific molecules and changes in the plasma membrane;the acrosome reaction (AR) is an exocytotic event induced by calcium influx, enabling the spermatozoa to penetrate the zona pellucida. These processes can be achieved only if the spermatozoa have good viability;indeed, determination of sperm viability is used for the assessment of semen quality. Membrane integrity and mitochondrial activity are important viability parameters of spermatozoa and fluorescent techniques based on membrane permeability to dyes have been developed to determine these parameters. The aim of this work was to determine the viability of boar sperm (fresh, one hour of capacitation induction and 20 min of AR induction) by flow cytometry using propidium iodide (PI) (1.25 μg/mL) and rhodamine 123 (R123) (0.20 μg/mL). Aliquots of 5 × 105 sperm were incubated with each fluorochrome separately and simultaneously for 10 or 20 min, respectively, at 38℃. The proportion of labeled spermatozoa and their fluorescence intensities were measured using a flow cytometer. The fluorescence index (FI) with PI gradually increased during the incubation and we found significant differences between all the groups. With R123, the FI increased in the capacitated sperm but decreased in the acrosome-reacted sperm, with significant differences between the fresh and capacitated spermatozoa. Our results suggest that the increase in the R123 fluorescence intensity in capacitated spermatozoa is due to changes in the mitochondrial membrane activity because the spermatozoa experienced changes in membrane fluidity and flagellar activation during capacitation. The use of fluorochromes and flow cytometry is a good tool for monitoring many markers of sperm function. Although capacitation and AR processes have been well studied, there is still much information to be elucidated with regard to these complex processes.

GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm.However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.

Bacteriospermia among smallholder artificial insemination boars in the Philippines and potential associated factors

Objective:To determine the prevalence of bacteriospermia,the bacterial load,and the potential factors associated with bacterial contamination in boar semen collected by local smallholder artificial insemination operators.Methods:Fifteen individual raw semen samples were collected from locally available artificial insemination boars owned by different smallholder boar operators within the 5th district of Leyte,Philippines and were subjected to standard bacteriological culture and identification,including a survey of potentially associated factors.Prevalence and bacterial count were determined accordingly,while boar characteristics and collection practices were clustered following agglomerative hierarchical clustering technique.Results:One hundred percent contamination with a bacterial count of(2.01±0.38)×10^(3) CFU/mL was observed.At least 73.33%of the samples were positive for Bacillus spp.,while other identified isolates included Enterobacter spp.,Staphylococcus spp.,E.coli,Pseudomonas spp.,Citrobacter spp.,and Klebsiella spp.Conclusions:Despite the high prevalence of bacteriospermia,the bacterial count is low.Nevertheless,on-farm practices on boar health and management,semen collection,and sanitation as well as the enhancement of basic protocols to control contamination should be conscientiously considered in smallholder artificial insemination operation.

Relationship between Serum L-Carnitine Levels and Sperm Parameters in Boars

This study evaluated the relationship between serum L-carnitine level and sperm parameters in young boars. Serum L-carnitine and semen characteristics were determined for 61 young Duroc boars between the ages of 590 and 630 days. Multiple linear regression analysis was performed to predict total and progressive motility and the total number of spermatozoa based on serum total L-carnitine and free L-carnitine levels. Total number of spermatozoa was not associated with basal serum L-carnitine levels. A regression equation was found in which both total L-carnitine levels and free L-carnitine levels were significant predictors of total and progressive motility (P 0.05). These results suggest that serum L-carnitine level is an important selection parameter for stock boars.

Effect of Stabilized Fish Oil Source on Sperm Quality and Production of Boars

Research findings for supplementing boar stud diets with fish oils are inconsistent. This study was designed to address three possible causes of performance variation of boars to fish oil supplementation: stability of the fatty acid source, level of inclusion and breed of boars tested. Three groups of 87 boars each, from two genetic lines (PIC 337 and PIC 800), were assigned to treatment based on age, mean sperm production (previous 12 weeks), and body condition score. All boars received a corn-soybean meal diet with a commercial fish oil supplement providing 1.83 g/boar/day of docosahexaenoic acid (DHA) as a preconditioning diet. On 10-Aug., 2020, the DHA source was changed to a stabilized starch imbedded source of refined fish oil (Salmate®), providing 1.83 g/b/d for the test diet. Two additional levels providing 2.38 and 2.94 g/b/d of DHA were fed for a 9 week pretreatment period and during the test period. Salmate® fed at 2.38 g/b/d of DHA resulted in a reduction in the number of rejected ejaculates (P < 0.045) by 7.5% and 6.4% compared to the lowest and highest inclusion rates, respectively. There were no treatments by genetic line interactions. A retrospective study of semen production and quality of 77 boars on the Salmate® diet containing 1.83 g/b/d DHA was done to compare to the original source of DHA at the same inclusion level. There were no differences in semen quality parameters between the 2 lipid sources. Ejaculate volume increased from 177.9 ml to 233.4 ml (P < 0.001) and total sperm cells per ejaculate increased from 69.7 × 109 to 82.0 × 109 (P < 0.001) due to substitution of Salmate®. Adding Salmate® at 2.38 g/b/d resulted in a lower number of rejected ejaculates per boar by 7.5% and 6.4% vs. 1.83 and 2.94 g/b/d, respectively, and boars fed Salmate® at 1.83 g/b/d produced 17% more doses than the competing product.

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super AQPs.AQP3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation.Water exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of sperm.However,the freezethaw process leads to sperm damage and a loss of fertilizing potential.Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen freezability.In this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability.Moreover,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

采精频率对长白种公猪精液品质的影响

为探讨采精频率对种公猪精液品质造成的影响,本试验采用计算机辅助精液分析仪检测了四川某种猪繁育场的10头长白种公猪在不同采精频率下(1、2、3、4、5次/周)的精液,分别其对精子活力、密度、精液量、精子结构变化和精子运动性能等指标的影响。结果表明:采精频率为3次/周时,精子密度最高,与采精频率为4次/周的精子密度差异不显著(P> 0.05),与采精频率为1、2和5次/周时的差异显著(P> 0.05);采精频率为3次/周时精子活力和活率最高,与采精频率为5次/周时的活力与活率差异显著(P> 0.05);精子总数在采精频率为3次/周时最多,精液量未呈现出规律性变化,但在采精频率为1次/周时最多,与采精频率为5次/周时的差异显著(P> 0.05)。综上所述,采精频率为3次/周时精液品质最佳;采精频率为4次/周和2次/周时精液品质次之。

猪精液冻存技术研究进展

猪精液冻存技术是提高生猪养殖业产值、降低养殖风险的关键辅助技术,也是育种和保种的基础技术,因此受到很大的关注,但在实际生产中,由于冻精与鲜精存在显著的品质差距,目前未获得广泛的应用推广。其关键难题在于猪精子质膜非极性相成分特殊、冷冻复苏后更易受到氧化损伤等。论文综述了猪冻精技术的应用现状、损伤机制的研究和改善手段,并介绍了抗氧化剂在冷冻复苏过程的添加策略以及利用蛋白质组学和寻找生物标志物来对冷冻复苏后猪精子受损的分子机制进行研究的可能性,以期为猪精液冷冻技术的进一步完善提供参考。

采精员和猪舍环境参数对杜洛克公猪精液品质的影响

【目的】研究采精员、猪舍温湿度、氨气浓度和光照强度对种公猪精液品质的影响,以提高猪精液品质。【方法】选取347头(580±48)d的杜洛克公猪,收集2021年11月至2022年2月共2966条精液测定记录,采精员做好每天公猪的采精记录,猪舍温湿度、氨气浓度和光照强度参数于每天上午09:00进行测定。使用R软件对采精员记录、环境参数和精液性状进行关联分析。【结果】①采精员对精液品质具有显著影响(P<0.05),8号采精员采集的精液精子活力最高、精子畸形率最低。②猪舍环境温度为22~25℃时,精子活力显著高于18.5~22℃(P<0.05),精子畸形率显著低于18.5~22℃(P<0.05)。③相对湿度在70%~77%时精子活力最高、精子畸形率最低,与相对湿度在46%~60%时差异显著(P<0.05),与相对湿度在60%~70%时无显著差异(P>0.05),相对湿度在60%~70%时精子活力、精子畸形率和总精子数都处于较高水平。④随着氨气浓度降低,精子活力和总精子数呈现上升趋势、精子畸形率呈下降趋势,氨气浓度2~4 ppm与5~7.3 ppm时精子活力、总精子数、精子畸形率差异显著(P<0.05)。⑤光照强度在90~110 lx时精子活力和直线前进运动精子比例最高,与光照强度为130~164.5 lx差异显著(P<0.05),光照强度对精子畸形率无显著影响(P>0.05)。【结论】猪舍环境参数温度范围为22~25℃、湿度范围为60%~70%、氨气浓度2~4 ppm和光照强度为90~110 lx时公猪的精液品质较高。生产中还应加强对采精员的管理培训,减少精液采集过程中造成的机械损伤及细菌污染,为今后研究、修订种公猪舒适环境参数,科学调控猪舍内环境提供参考和理论依据。

维甲酸对体外热处理猪精子活力、抗氧化性能及其糖脂代谢的影响

旨在探究维甲酸(RA)对体外热处理猪精子活力、抗氧化性能及其糖脂代谢的影响。将相同来源的精液离心重悬于EBSS培养基,随机分为5组,在39℃环境条件下分别孵育0、1、2、3和4 h后,测定精子活力,筛选合适的孵育时间。将相同来源精液离心重悬后,随机分为6组:对照(CON)组、10、50、100、1000 nmol·L^(-1)RA组和DMSO组,在39℃环境条件下孵育3 h,测定精子活力,筛选RA适宜添加剂量。将相同来源精液离心重悬后,随机分为4组:37℃、37℃+RA、39℃、39℃+RA。37℃+RA组和39℃+RA组培养基中RA浓度为100 nmol·L^(-1),37℃组和39℃组培养基中不含RA,37℃组和37℃+RA组于37℃环境下孵育,39℃组和39℃+RA组于39℃下孵育,测定精子活力,并利用蛋白荧光免疫方法,检测维甲酸受体α(RARα)蛋白定位表达,收集精子用于精子抗氧化和糖脂代谢指标的测定。结果显示:1)39℃处理条件下,随着孵育时间的增加,精子的运动性能逐渐变差。2)39℃处理3 h条件下,RA能改善精子运动性能,且浓度为100 nmol·L^(-1)时效果最好。3)与37℃组相比,39℃培养会显著降低精子总活力及前进运动精子比例(P<0.05),显著提高不运动精子比例(P<0.05);与39℃组相比,培养基中含100 nmol·L^(-1)RA能显著提高精子总活力、前进运动精子比例以及快速运动精子比例(P<0.05),显著降低不运动精子比例(P<0.05);与37℃组相比,39℃培养能显著增加精子丙二醛(MDA)水平(P<0.05),降低T-SOD活力(P<0.05);与39℃组相比,培养基中含100 nmol·L^(-1)RA能显著降低精子MDA水平(P<0.05),提高精子T-SOD活力(P<0.05)。39℃培养和RA处理对精子丙酮酸、葡萄糖、总胆固醇及甘油三酯含量无显著影响。4)RARα蛋白表达主要集中在精子尾部的主段和末段,头部和富含线粒体的中段有较弱表达,但不明显。100 nmol·L^(-1)RA未明显改变RARα蛋白表达位置。结论:猪精子能够表达RARα蛋白,热处理使精子运动性能显著降低,RA能改善热处理猪精子的运动性能,这可能与RA能降低精子MDA含量,增加T-SOD活性,提高精子抗氧化能力有关。

大白公猪精浆外泌体miRNA表征鉴定及功能分析

[目的]精浆外泌体(Seminal plasma exosome,spEX)在精子成熟、凋亡、受精中起着至关重要的作用。本文旨在探究种公猪spEX miRNA表达及miRNA在精子成熟及功能维持过程中的潜在调控作用。[方法]提取大白公猪spEX并进行电镜分析、粒径分析、标志性蛋白表达分析和miRNA高通量测序。[结果]成功分离出spEX,利用miRNA测序共鉴定出329个spEX miRNA。对高表达miRNA进行靶基因预测和功能富集分析,结果表明spEX miRNA在射精、P53信号通路、前列腺癌、细胞对DNA损伤刺激的反应、负调控凋亡过程、顶体膜结合、受精等通路中均发挥了潜在调控作用。[结论]本研究为spEX miRNA在调控精子活力和精子受精作用方面提供基础数据,并为精液保存调控机制的研究提供参考。

富硒益生菌对种公猪精液品质的影响

在成年长白种公猪的饲料中,以0.3 mg/kg(以硒浓度计算)添加富硒益生菌,研究了富硒益生菌对种公猪精液品质的影响。结果显示,试验组种公猪精液中的精子活率、顶体完整率、还原型谷胱甘肽过氧化物酶的活性均比对照组高,但精液中的精子畸形率比对照组低,说明富硒益生菌能明显改善种公猪的精液品质。

温度和保存时间对猪精子顶体酶活性的影响

为了探讨温度和液相保存时间对猪精子顶体酶活性的影响,采用紫外分光光度计检测猪精子顶体酶活性。结果显示:(1)鲜精、冷休克和热休克每106精子顶体酶活性分别为5.38、5.20和5.16 mIU,差异不显著(P>0.05)。(2)鲜精在4、17、25、30、37和39℃时每106精子顶体酶活性分别为4.29、5.01、5.03、5.17、4.78和4.61 mIU,随着温度的升高精子顶体酶活性逐渐提高,当温度达到30℃时达到最高,且4℃和30℃时差异显著(P<0.05)。(3)液相精液在4℃保存超过1 d时精子顶体蛋白酶活性显著下降(P<0.05);在17、25和39℃保存超过2 d时顶体酶活性显著下降(P<0.05),且在17℃保存效果较好。结论是猪精子顶体蛋白酶活性受温度的影响,在17℃的条件下3 d内保存的猪精子顶体酶活性最佳。

稀释液中添加海藻糖对猪冷冻精液质量参数和活性氧的影响

本试验旨在探讨海藻糖对猪精子冷冻效果以及精子内活性氧(ROS)的含量影响。试验分对照组和4个海藻糖处理组(0.025,0.05,0.1和0.2 mol/L)。精子冷却后,添加0.1 mol/L海藻糖处理组精子活力显著(P<0.05)高于对照组,而精子内和稀释液中各组间ROS发生量都没有显著的差异。精子冷冻解冻后,添加0.05 mol/L海藻糖处理组的精子活力显著(P<0.05)高于对照组;添加0.05 mol/L和0.1 mol/L海藻糖处理组的精子生存率显著(P<0.05)高于对照组;添加0.1 mol/L海藻糖处理组顶体完整的精子百分比显著(P<0.05)高于对照组;4个海藻糖处理组膨胀精子百分比都显著(P<0.05)高于对照组;添加0.025 mol/L海藻糖处理组ROS水平显著(P<0.05)低于对照组。结果表明:海藻糖对精子冷冻保存是有益的,且能抑制精子内ROS的发生,但它的作用机制有待于进一步的研究。

海藻糖对猪精液冷冻保存效果的影响

在传统的Tris-柠檬酸-葡萄糖稀释液基础上,分别添加25%、50%、75%、100%的海藻糖,研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明,海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果,其最佳添加浓度为25%,冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高(P<0.05),分别达到41.38%、46.34%、44.56%、43.51%和64.09%。海藻糖可以明显抑制精子获能,获能处理前精子获能率仅为3.68%,而获能处理后达到41.82%,有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2%,海藻糖只有与甘油共同作用,才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系(P<0.01),而与获能处理前精子的获能率存在显著的负相关关系(P<0.05)。

左旋肉碱类营养液对猪精液活力的影响

以大白猪和长白猪为研究材料,对照组不饲喂左旋肉碱类营养液,2个处理组分别饲喂25和30 ml左旋肉碱类营养液,研究左旋肉碱类营养液对猪的原精活力和精子稀释活力的影响。结果发现:饲喂左旋肉碱类营养液的处理组,种公猪的原精活力和精子稀释活力均极显著高于对照组(P<0.01),而同品种内2个处理组间原精活力和精子稀释活力差异不显著(P>0.05)。

单细胞电泳技术检测低温保存猪精子的DNA损伤

应用单细胞凝胶电泳技术（SCGE），对冷冻解冻后猪精子DNA的损伤情况进行研究。结果表明，冷冻解冻会对猪精子DNA造成损伤，其彗星率与鲜精相比差异显著（P〈0．05）；各组冻精之间，甘油添加量为2％～3％（v／v）时，冷冻对猪精子DNA造成的损伤最低，彗星率均低于其余各组（P〈0．05）。

猪精子冷冻技术的研究

采用手握法采集健康成年的长白公猪猪精子,室温离心后,采用液氮熏蒸法细管冷冻猪精子。以精子活力为判定指标,比较了平衡时间和甘油浓度对猪精子冷冻的影响。结果表明:(1)Ⅱ组精子冷冻复苏后精子活力(35.83±5.38)%要高于其他组(P<0.05);(2)Ⅰ组和Ⅱ组冷冻复苏精子的活力[(29.75±4.38)%,(28.40±8.55)%]要高于其他组(P<0.05),由此得出最佳平衡时间和甘油浓度。