Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from Yorkshire, Hampshire, and Landrace boars.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.     

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between −4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs.     

Semen from boars infected with porcine reproductive and respiratory syndrome virus (PRRSV) contains antibodies against structural as well as nonstructural viral proteins

The seminal excretion of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) was examined in a group of five boars experimentally infected by the nasopharyngeal route. By using phage-displayed peptide epitopes from the PRRSV replicase and envelope glycoproteins as ELISA antigen, we were able to separately and specifically assay antibody responses against structural and nonstructural viral proteins. Antibodies against structural as well as nonstructural viral proteins were consistently found in the semen of all boars, beginning from 1-4 weeks postinfection. This is the first report documenting the presence of anti-PRRSV antibodies in boar semen. Seminal antiviral IgA was also detected, and we observed a correlation between seminal IgA responses against nonstructural viral proteins, and the duration of PRRSV RNA excretion in semen. The implications of these findings for the diagnostics and pathogenesis of venereal PRRSV infection are discussed.     

种公猪精液中猪瘟和蓝耳病病毒混合感染的快速检测

参考GenBank公布的猪蓝耳病病毒(PRRSV)VL2332株、LV株以及猪瘟兔化弱毒(CSFV)C株的基因序列,各设计合成了一对引物,建立了在相同PCR扩增条件下能同时检测PRRSV和CSFV的RT-PCR方法。对2003~2004年期间江苏、浙江、安徽、福建、上海等省市的17个大中型猪场送检的186份种公猪精液进行了检测,结果18份呈PRRSV阳性,24份呈CSFV阳性,其中有11份为PRRSV和CSFV的混合感染,约占送检精液样品的5.91%。试验结果表明,所建立的RT-PCR方法可用于精液中这2种野毒感染的快速鉴定和分子流行病学调查。     

骨桥蛋白在长白公猪生殖细胞中的表达及其与繁殖性能的相关性

本试验旨在研究骨桥蛋白（osteopontin,OPN）在长白公猪生殖细胞中的表达和定位,并探究其与公猪繁殖性能的相关性。试验采集了长白公猪精液和不同阶段（3日龄、3月龄、6月龄和12月龄）的睾丸组织,通过蛋白印迹的方法检测OPN蛋白在精液和不同月龄睾丸中的表达,通过免疫组化的方法对OPN蛋白在公猪睾丸细胞中进行定位;同时,根据配种胎次≥ 20胎,3次配种公猪为同一头的标准,筛选并采集17头长白种公猪精液,统计相对应的1 388头母猪的生产成绩,计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。低温离心精液分离得到精子和精浆,丙酮法提取精浆蛋白,Lysis buffer方法提取精子蛋白,最后运用BCA和ELISA的方法检测精子和精浆中OPN蛋白的含量,分析OPN蛋白与公猪繁殖性能的相关性。蛋白印迹结果显示,OPN在精子、精浆和各月龄阶段的长白公猪睾丸中均以两种形式表达（67.4和33.7 ku）,且67.4 ku的形式在3月龄公猪睾丸中表达量最高;免疫组化的结果显示,OPN在长白公猪的初级精母细胞、次级精母细胞和精子细胞中表达,在精母细胞、支持细胞和间质细胞中无表达;BCA和ELISA结果显示,精子中的OPN蛋白含量是精浆中的7倍（P<0.05）,精液中的OPN蛋白与公猪窝产活仔数显著正相关（P<0.05）。本试验结果表明,OPN在各阶段的长白公猪睾丸中都有表达,且在精子和精浆中也有表达,这可能与公猪的繁殖性能有关,从而为后期OPN蛋白在公猪受精力的研究奠定基础。     

Transmission of agents of the porcine respiratory disease complex (PRDC) between swine herds: a review. Part 2--Pathogen transmission via semen, air and living/nonliving vectors

The transmission of PRDC-pathogens (PRRSV, influenza virus A, PCV2, M. hyopneumoniae, A. pleuropneumoniae) between swine herds, which was summarized in the first part of the review, mainly occurs via pig movement. The risk of pathogen transmission by insemination with contaminated semen plays only a relevant role in the infection with PRRSV and PCV2. A risk of the aerogen transmission of pathogens between herds within a distance of 2 to 3 km is described for M. hyopneumoniae and PRRSV. Evidence for the other pathogens is not investigated. The PRDC-pathogens are frequently detected in wild boar populations. Therefore, the transmission between wild boars and domestic pigs seems possible by close contacts. PRRSV and M. hyopneumoniae can be transmitted by contaminated clothes and boots, but the use of sanitation protocols appears to limit their spread. Live vectors like rodents or birds seemed to have no special importance for the transmission of PRDC-pathogens.     

Nested polymerase chain reaction detection and duration of porcine circovirus type 2 in semen with sperm morphological analysis from naturally infected boars.

A nested polymerase chain reaction (nPCR) protocol was applied to porcine semen to demonstrate the porcine circovirus type 2 (PCV2) shedding patterns and duration in naturally infected boars. Sperm morphology analysis was performed on a subset of samples to determine if the presence of PCV2 DNA in semen was associated with reduced semen quality. Semen was collected serially from 43 boars representing 6 breeds, aged 33.9 to 149.3 weeks. Of the 903 semen samples collected, 30 samples (3.3%) were positive for PCV2 DNA by nPCR from 13 boars. Boars shedding PCV2 DNA in semen ranged between 35.9 and 71.0 weeks of age, and shedding occurred during a period of up to 27.3 weeks. A semen nPCR test was 2.6 times more likely to be positive when collected from pigs that were < or =52 weeks of age, and 3.0 times more likely to be positive when collected from pigs that were < or =26 weeks from time of entry into the stud main unit (generalized estimating equations: P = 0.02; 95% confidence interval [CI] of the odds ratio 1.2 to 5.5, and P = 0.01; 95% CI of the odds ratio 1.3 to 6.9, respectively). These results demonstrate a sporadic and long-term shedding pattern of PCV2 DNA in semen from naturally infected boars. PCV2 DNA in semen does not appear to have detrimental effects on sperm morphology; however, boar age and, possibly, breed may contribute to the persistence of PCV2-shedding in semen.     

Adjusted method of penis fixation during boar semi-automatic semen collection aiming to reduce bacterial contamination

Semen collection has an essential role in the initial bacterial load in boar ejaculates and extended semen. The study aimed to explore the efficacy of an adjusted penis fixation in a semi-automatic collection system on reducing bacterial contamination of ejaculates in two-boar studs with different scenarios. Historically, stud A had low levels of bacterial load in raw semen, while stud B had a high level of contamination. A total of 56 mature boars had their semen collected using two methods of penis fixation: (a) Traditional: The penis was fixed directly with the artificial cervix and transferred to the adjustable clamp; (b) Adjusted: The fixation was performed with one gloved-hand, and after exteriorization, the penis was gripped using the artificial cervix with the other gloved-hand and transferred to the adjustable clamp. The bacterial load (p = .0045) and the occurrence of ejaculates >231 CFU/ml (p = .0101) were reduced in the Adjusted compared to the Traditional method. Bacterial load was reduced when using the Adjusted method in stud B (p = .0011), which showed a greater occurrence of critical factors for bacterial contamination (p ≤ .0034). The Adjusted method reduced the occurrence of ejaculates >231 CFU/ml when the preputial ostium was dirty (p = .016) and the duration of semen collection was >7 min (p = .022) compared to the Traditional method. In conclusion, the Adjusted penis fixation was efficient in reducing bacterial load of ejaculates, mainly in boar stud B, which had high contamination challenges.     

种公猪精液伪狂犬病野毒的筛查

本文基于1 100头基础母猪的规模化场进行公猪精液伪狂犬病病毒筛查,农业部规定伪狂犬病病原学检测方法是通过ELSIA方法检测猪伪狂犬病gE野毒抗体。由于抗体检测具有滞后性,导致正在发生感染的猪群g E抗体检测往往呈阴性,但此时通过病原学检测精液已经正在排毒。本文互补了ELSIA抗体检测方法与荧光定性PCR检测病原的优劣,成功实现种公猪精液伪狂犬病病毒的筛查。     

猪精液中繁殖与呼吸综合症病毒的抗性药物筛选研究

猪蓝耳病毒等在感染的猪的精液中可检测到相关病毒,能过精液导致疫病爆发已有报道,筛选在体外精液中抗病毒药物组方意义重大.应用细胞培养和病毒滴度试验,比对筛选不同类型抗病毒中药、化药及组合配方抗猪精液中蓝耳病病毒（PRRSV）的作用效果.结果表明黄芩、利巴韦林、银黄与利巴韦林组合制剂3种配方具有抗猪精液中蓝耳病病毒（PRRSV）的作用,其中银黄和利巴韦林组合配方对蓝耳病病毒（PRRSV）抑制率达到78％,抗猪精液中蓝耳病病毒（PRRSV）效果显著.为人工授精和冷冻精液制贮备中预防疫病发生提供了基础.     

Do CASA systems satisfy consumers demands? A critical analysis

Boar studs are often offered new technologies including several CASA (computer-assisted semen analysis) systems. However, independent information to assist their purchase decisions is not available. The systems accuracy and repeatability variation because of different factors can be evaluated through duplicate testing of semen samples and comparison of the results according to WHO standards for humans. This primary analysis and a thorough economic cost benefit evaluation will help to decide whether the purchase of a CASA system will be profitable for a boar stud. Our experience of implementing several CASA systems in the cooperative Dutch Artificial Insemination (AI) centres is used as a base for this discussion.     

品种、季节、胎次、采精月龄及采精间隔对猪精液品质的影响

【目的】探究品种、公猪出生胎次、公猪同窝仔猪数、公猪乳头数、采精季节、采精月龄和采精间隔对猪精液品质的影响,以及不同品种对精液质量稳定性的影响。【方法】选取909头杜洛克、长白、大白种公猪为试验群体,收集2021年4月至2022年4月27 408条精液测定记录,采用混合线性分析模型和方差分析探究品种、公猪出生胎次、公猪同窝仔猪数、公猪乳头数、采精季节、采精月龄和采精间隔对精液体积、精液密度、精子活力、直线前进运动精子比例、精子畸形率、总精子数及各精液性状稳定性的影响。【结果】从不同品种对精液品质的影响来看,长白猪精液体积和总精子数均显著高于大白猪、杜洛克猪(P＜0.05),杜洛克猪精液密度显著高于大白猪和长白猪(P＜0.05),杜洛克猪、大白猪精子活力均显著高于长白猪(P＜0.05),大白猪直线前进运动精子比例显著高于长白猪和杜洛克猪(P＜0.05),长白猪和大白猪精子畸形率均显著低于杜洛克猪(P＜0.05);从不同公猪出生胎次对精液品质的影响来看,1~3胎出生的公猪具有较高的精液品质;从不同采精季节对精液品质的影响来看,精液密度、精子活力和总精子数秋、冬季显著高于春、夏季(P＜0.05);从不同采精月龄对精液品质的影响来看,16~25月龄公猪具有较高的精液品质;从不同采精间隔对精液品质的影响来看,4~5 d为最佳采精间隔,采精间隔过长会导致精子畸形率上升;从不同公猪总乳头数来看,乳头数13~16个时,各精液性状品质都处于中等水平,有利于公猪生产应用。3个品种各精液性状间稳定性趋势不一,杜洛克猪和大白猪精液体积和精子活力的稳定性均显著高于长白猪(P＜0.05),杜洛克猪精液密度、精子畸形率和总精子数的稳定性均显著高于大白猪和长白猪(P＜0.05),大白猪直线前进运动精子比例的稳定性显著高于长白猪和杜洛克猪(P＜0.05)。各品种中,长白猪稳定性较差;各精液性状中,精子活力稳定性最好。【结论】品种、公猪出生胎次、采精季节、采精月龄和采精间隔均会影响公猪精液品质,可根据不同品种公猪制定更完善的选择方案,提高精液质量,加速公猪遗传改良。     

Effect of vaccination of boars against porcine circovirus type 2 on ejaculate characteristics, serum antibody titers, viremia, and semen virus shedding

The objective of this research was to determine the effect of vaccination against porcine circovirus type 2 (PCV2) on ejaculate characteristics, PCV2-specific antibody titers in serum, viremia, and viral shedding in the semen of PCV2-positive boars. Before vaccination, all boars were confirmed by PCR to be naturally infected with PCV2. The boars were vaccinated with a commercial killed vaccine against PCV2 (n = 5) or served as controls and received 2 mL of 0.9% saline (n = 5). Semen and blood samples were collected before vaccination at wk 0 and at 7-d intervals thereafter until wk 8. Sperm concentration and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system, and sperm morphology was evaluated using light microscopy after staining. The PCV2 antibody titers were determined in serum using an ELISA, and the genomic copy numbers of PCV2 DNA in serum and semen were determined by real-time PCR. In general, there were no effects of treatment or treatment × week on semen or sperm characteristics (P > 0.10). An effect of treatment × week was detected for serum antibody titers (P < 0.01). Compared with controls, PCV2 antibody titers in vaccinated boars were less (P < 0.01) at wk 7 (1.01 ± 0.05 titer/mL vs. 1.23 ± 0.05 titer/mL) and tended (P = 0.07) to be less at wk 8 (1.05 ± 0.05 titer/mL vs. 1.17 ± 0.05 titer/mL). There were no effects of treatment or treatment × week for serum and semen genomic copy numbers of PCV2 DNA (P > 0.10). There was a tendency (P = 0.09) for an effect of week on serum viral load. It was evident that during this experiment, boars experienced reoccurring PCV2 infection, and the detection of an increased PCV2 DNA load in serum preceded that in semen; the duration of reoccurring infection appeared to be less in vaccinated boars compared with controls. In summary, vaccination against PCV2 can reduce antibody titers when given postinfection and has no dramatic effect on indicators of semen quality. Vaccination against PCV2 in naturally infected boars can also decrease the length of reoccurring infection and decrease the duration of viral shedding in semen.     

公猪营养与繁殖

公猪的重要性越来越得到广泛的关注。尽管种公猪在猪群中占据比例不高,但是种公猪的繁殖性能可以直接影响母猪的妊娠率和产仔数,进而影响猪场的整体经济效益。公猪的精液品质是其繁殖力的基础,同时也是发挥公猪遗传性能的重要保障。文章综合了近年来国内外研究公猪营养对精液品质及繁殖力的调控,从膳食纤维、蛋白质及氨基酸、多不饱和脂肪酸、微量元素及维生素、植物提取物、有益菌群等方面分析了它们如何改善公猪精液品质,但是有关研究还很不完善、不系统。因此,需要根据种公猪不同品种、不同生长阶段、不同环境条件下对各类营养物质的需求来制定合理、合适的种公猪饲料配方,以期发挥种公猪的最大潜力和价值。     

不同品种、月份、月龄及采精间隔对猪精液品质的影响

为研究不同品种、采精月份、采精月龄和采精间隔等因素对加系公猪精液品质的影响,以及品种、初次采精周龄对精液质量稳定性的影响,本研究以江西某种公猪站79头加系大白猪、长白猪、杜洛克猪种公猪为试验群体,收集2018年12月至2020年12月3 921条精液采集与精液质量数据,通过混合线性模型与方差分析探究各因素对精液量、精液密度、精子活力、总精子数及其稳定性的影响。结果显示,从不同品种对精液质量的影响来看,长白猪精液量和总精子数均高于大白猪、杜洛克猪,但杜洛克猪精液密度高于长白猪、大白猪,杜洛克猪精子活力最低;从不同月份来看,1~3月采精精液密度最高,4~6月采精精子活力最高,10~12月采精精液量和总精子数最高,精液量呈现秋冬多、春夏少的季节变化规律。公猪不同月龄采精,精液质量指标也存在差异,月龄越小精液量越低,但精液密度偏高,精子活力相对较好,在19~24月龄黄金期总精子数最高。不同采精间隔对精液质量有较大影响,采精间隔越长,精液量、精液密度、精子活力和总精子数相对较好,采精间隔为5 d时综合性能最佳,但过长的采精间隔导致精子活力降低。品种影响总精子数稳定性,长白猪、大白猪总精子数稳定性显著优于杜洛克猪（P<0.05）。本研究结果表明,品种、采精月份、采精月龄和采精间隔均会影响公猪精液质量,关注这些因素有助于公猪站制定更完善的生产计划,提高公猪利用率。     

GPx6在大白猪附睾细胞中的表达及其与繁殖性能的相关性

本试验旨在研究谷胱甘肽过氧化物酶6（glutathione peroxidase 6,GPx6）在大白公猪附睾细胞中的表达和定位,并探究其与公猪繁殖性能的相关性。选取15月龄的公猪和母猪各3头,取睾丸、附睾、前列腺、尿道球腺、精囊腺、卵巢和输卵管,提取蛋白,通过蛋白免疫印迹和免疫组化（IHC）检测组织和细胞中GPx6蛋白的表达和定位;根据评定大白公猪受精能力的数学模型,按照公猪配种胎次≥20胎、3次配种公猪为同一头的标准,筛选并采集符合要求的20头公猪精液,同时,统计相对应的1 279头母猪的生产成绩,计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。提取精子蛋白和精清蛋白,通过BCA和ELISA检测精子和精清中GPx6蛋白的含量。使用SPSS软件的独立样本t检验及单因素方差分析（one-way ANOVA）,进行差异显著性分析,用双变量Pearson进行相关性分析,P<0.05表示差异或相关显著。结果表明,GPx6蛋白在附睾中高表达,IHC结果显示,GPx6蛋白在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,在肌样细胞中不表达;精清蛋白中GPx6的含量是精子蛋白的7倍,精液中GPx6蛋白的含量与窝产活仔数、窝产总仔数呈负相关关系。结果提示,GPx6在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,且其在精液中的含量会影响公猪的繁殖性能,这为GPx6对公猪受精能力影响的研究奠定了理论和试验基础。     

Effect of vitamin supplements on some aspects of performance, vitamin status, and semen quality in boars

The aim of the present study was to determine the effects of dietary supplements of vitamins on vitamin status, libido, and semen characteristics in young boars under normal and intensive semen collection. Sixty Landrace, Yorkshire, and Duroc boars were allocated randomly from 6 to 10 mo of age to one of the following diets: 1) basal diet (industry level) for minerals and vitamins (Control, n = 15); 2) basal diet supplemented with vitamin C (ASC, n = 15); 3) basal diet supplemented with fat-soluble vitamins (FSV, n = 15); and 4) basal diet supplemented with water-soluble vitamins (WSV, n = 15). After puberty (approximately 12 mo of age), semen was collected at a regular frequency (three times every 2 wk) for 5 wk. Thereafter, all boars were intensively collected (daily during 2 wk). A recovery period (semen collection three times every 2 wk) followed and lasted for 10 wk. Sperm quality (percentage of motile cells and percentage of morphologically normal cells) and quantity (sperm concentration, semen volume, and total sperm number) were recorded as well as direct and hormone related measurements of boar libido. Blood and seminal plasma samples were taken to monitor vitamin status. High concentrations of B6 (P < 0.05) and folic acid (P < 0.05) were observed in the blood plasma of WSV boars, whereas greater concentrations of vitamin E (P < 0.01) were obtained in FSV boars. In the seminal plasma, folic acid concentrations tended to be greater in WSV boars (P < 0.08). During the intensive collection period, there was a tendency (P < 0.06) for semen production to be greater in WSV boars, the effect being less pronounced (P < 0.10) in FSV boars. During the recovery period, the percentage of motile sperm cells was greater in WSV boars (P < 0.03) and, to a lesser extent, in FSV boars (P < 0.10) compared with Control boars. Sperm morphology and libido were not affected by treatments. These results indicate that the transfer of vitamins from blood to seminal plasma is limited and the dietary supplements of water-soluble and fat-soluble vitamins may increase semen production during intensive semen collection.