Studies on boar semen. III. Sperm concentration and seminal plasma total solids followed in Danish AI boars through a 10-year-period

Semen samples from 1531 young boars eligible for AI service were examined for normality. Sperm concentration (s.c.) was determined by the hemocytometer technique in 1348 of the samples. Seminal plasma total solids (t.s.) were determined on all samples in order to control whether the semen samples originated from complete ejaculates.The hemocytometer counts showed an arithmetic mean of 370 × 10⁶/ml with a standard deviation of 188×10⁶/ml. No correlation was found either between s.c. and age or between s.c. and season of the year.The seminal plasma t.s. showed an arithmetic mean of 4.6% with a standard deviation of 1.35%. No correlation was found between t.s. and s.c. or between t.s. and the age of the boars. Neither was there any association between t.s. and the season of the year.Values of t.s. below 1.6% combined with aspermia were regarded as the result of incomplete ejaculations and the following retest from such cases gave ejaculates showing normal values concerning s.c. and t.s. A drop in t.s. combined with an admixture of pathological cells to the ejaculate may indicate an inflammatory condition in the vesicular glands.     

冷冻保存对公猪精子功能的影响

试验旨在探究公猪精液冷冻保存对其精子功能的影响。取长白猪的鲜精和优质冻精,用精子分析仪检测精子的运动能力,台盼蓝染色检测精子活率,体外受精（IVF）试验检测卵裂率与囊胚率,采用不同功能检测试剂盒检测冻精和鲜精的顶体完整率、线粒体膜通道孔（MPTP）活性、线粒体膜电位（MMP）、线粒体活性、线粒体氧化应激活性氧（ROS）以及精子DNA完整性,实时荧光定量PCR检测弱精子症相关蛋白基因SMCP、TEKT3、DNAH1、TCTE3的表达。结果表明,与猪鲜精相比,猪冻精的活率及活力均显著降低（P<0.05）,冻精的顶体完整率也明显下降（P<0.05）;冻精的卵裂率和囊胚率显著低于鲜精（P<0.05）;精子线粒体功能分析结果显示,冻精的MPTP相对荧光单位值（RFU）、线粒体膜电位荧光比率以及线粒体活性光密度（OD）值均显著低于鲜精（P<0.05）;精子线粒体ROS检测发现,冻精的RFU值显著高于鲜精（P<0.05）;精子DNA完整性检测结果显示,冻精拖尾率显著高于鲜精（P<0.05）;而弱精子症相关蛋白基因的表达与鲜精相比,差异不显著（P>0.05）。综上所述,冷冻导致猪精子活率、活力、线粒体功能、DNA完整性下降,最终使得冷冻精液精子的受精能力降低。     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

冬夏季长白猪精液中细菌多样性与精子质量特性关联分析

为探究冬夏季长白猪精液中细菌多样性变化和精子质量特性之间的相关性,利用16S rRNA基因高通量测序技术得到冬夏季长白猪精液中微生物的特征序列(ASV),计算机辅助精子分析系统(CASA)分析冬夏季精子活力参数指标的变化.结果 显示:长白猪精液中细菌多样性在冬夏季之间差异显著(P＜0.05),门水平冬季优势菌群为厚壁菌...     

Method agreement between three different chambers for comparative boar semen computer‐assisted sperm analysis

The computer‐assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 10⁶ sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 μm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non‐progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.     

Red‐light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED‐based red light increases its reproductive performance following artificial insemination (AI). Red‐light stimulation with MaXipig^® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red‐light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red‐light stimulation. Our results indicate that a routine use of red‐light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.     

猪精子顶体素水平与精液质量参数和体外受精间的联系

本研究目的是评估不同处理对精子顶体素活性的影响以及顶体素活性、精液参数和体外受精间的联系.鲜精、稀释精液(1:1)和液相精液(17℃)的精子顶体素活性分别是5.27,4.28和4.41 mIU/106.冻精或液相精液保存4 d后顶体素活性显著下降(P＜0.05).公猪间液相精液顶体素活性和低渗膨胀精液百分数有显著的差异(P＜0.05).冷、热应激对精子顶体素活性没有影响.鲜精和冻精的顶体素活性与低渗膨胀精子百分数和顶体完整率呈正相关.不同种猪液相精液精子顶体素活性与体外受精率和随后的囊胚发育率相关性不大.因此建议顶体素活性与其它精子功能参数相结合才能精确预测精子受精力.     

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐SM and 15‐SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5‐SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐SM and 15‐SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5‐SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.     

Identification of sperm morphometric subpopulations in cooled‐stored canine sperm and its relation with sperm DNA integrity

The aims of this study were to (i) identify different morphometric subpopulations in cooled‐stored canine sperm and their patterns of distribution during cool‐storage for up to 240 hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer‐assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72 hr and thereafter sP3 increased progressively. sDFi increased after 48 hr of cool‐storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool‐storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.     

Analysis of testicular variables,semen motility and kinematics-derived indexes in boar using a CASA-Mot system

The present study aimed to evaluate differences in area, volume and testicular weight among groups related to breed and age of boar and establish associations between testicular parameters and seminal characteristics. Seminal quality has been studied using computer-assisted semen analysis (CASA) systems that reduce the subjectivity of kinematic parameters. Based on the data obtained through this system, sperm quality indexes and their derived parameters have been assessed to identify a better estimator for determining movement and velocity parameters. We also established indexes of velocity and sperm movement for boar semen. To this end, we evaluated 191 ejaculate samples from 63 boars. Differences related to the effects of season and age on quality boar semen were found (p ≤ .05) in the animal groups. Indexes were established for velocity and sperm movement based on CASA-Mot, and we proposed a sperm movement index (SMI) and sperm velocity index (SVI) with the CASA-Mot variables. Correlations were found (p < .05) among sperm indexes and CASA-Mot variables. Boar characteristics were found to be associated with some CASA-Mot variables, and the estimated SMI and SVI indexes predicted a greater variation in kinematic characteristics compared to the parameters evaluated separately using the CASA-Mot system.     

Supplementation of salvianic acid A to boar semen extender to improve seminal quality and antioxidant capacity

The purpose of this test was to investigate the effect of salvianic acid A (SAA, CAS No. 76822‐21‐4) on the quality of boar semen during liquid storage at 17°C. The effects of different concentrations of SAA on semen quality and antioxidant capacity were analyzed. Boar semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 15, 30, 45, 60, 75 μM of SAA). During the storage period, sperm activity was measured every 24 hr, and plasma membrane integrity, acrosome integrity, total antioxidant capacity (T‐AOC), malondialdehyde (MDA) content, and catalase (CAT) activity were measured at 0, 1, 3, and 5 days. The results from our study suggest that different concentrations of SAA have different effects on semen preservation. Semen samples supplemented with SAA showed reduced effects of oxidative stress on sperm compared to the control samples. Supplementation of 30 μM of SAA significantly improved sperm motility, plasma membrane integrity, acrosome integrity, and antioxidant capacity. However, the addition of SAA to the extender was scarcely beneficial to the improvement of results of artificial insemination with boar semen after liquid preservation. Further studies are necessary in order to demonstrate that SAA has good effects on the liquid preservation of semen.     

Mobility of Japanese quail spermatozoa and its relationship to egg fertility

Sperm mobility (SM) appears to be primary determinant of fertility in chicken and turkey. The aims of this study were to extend the concept to the Japanese quail by developing an assay to quantify SM, explaining the basis of SM using motility properties measured by CASA, and exploring the relationship between SM and egg fertility. The study was carried out in three stages: i) males (n = 20) and females (n = 20) were mated individually; ii) ejaculates were collected from 20 males, and SM was measured; iii) males (n = 20) and females (n = 20) were mated individually. In Stages I and III, data were collected for egg fertility, Sperm_OPVL and Holes_IPVL. In Stage II, SM assay was developed and assay conditions were defined: effect of sperm numbers on absorbance in Accudenz solution; effect of Accudenz concentration on sperm motility and mobility; effect of quail proctodeal gland foam extract and incubation temperature on SM at 37 and 41°C. The recorded absorbance of sperm movement was dependent on sperm numbers in the sperm suspension overlaying the Accudenz (p < .001). At 41°C, SM, progressively motile sperm, VCL, VSL and VAP were negatively affected by Accudenz concentration (p < .05). The effect of foam on SM and motility depended on an interaction between the concentration of foam extract and incubating temperature. Males were categorized into low, average and high SM phenotypes. These categories differed significantly (p < .001), but sperm motility and SM were not related to egg fertility. In conclusion, SM assay can be used to identify mobility phenotypes, but the poor relationship between SM and egg fertility indicates a need for further studies on interaction between the concentration of foam extract, incubating temperature, and in vivo sperm movement and egg fertilization success.     

奶牛性控与常规冻精DNA损伤的彗星检测

本文为确定性控冻精与常规冻精的DNA损伤程度,利用彗星实验法分别将奶牛的性控冻精和常规冻精凝胶混合液在裂解液中作用后,使用普通载玻片双层铺胶,电泳,吖啶橙染色后在荧光显微镜下检测、拍照,经CASP软件图像分析后用SPSS 11.5进行数据统计,初步建立了奶牛精子DNA损伤的5级彗星图谱。统计结果显示,性控冻精的DNA损伤系数为116,显著高于常规冻精的损伤系数（28）（P〈0.01）;彗星率为63.06%,显著高于常规冻精的彗星率（31.13%）（P〈0.01）。可以得出,性控冻精DNA损伤显著高于常规冻精。     

A Bayesian analysis of boar spermatozoa kinematics and head morphometrics and their relationship with litter size fertility variables

The semen movement and sperm head size patterns of boar ejaculates were analysed using computer-assisted semen analysis (CASA)-Mot and -Morph systems. The aim of the present study was to compare morphometric and kinematics variables from boars and to determine the relationship with sow fertility variables related to litter size. The females were from maternal crossing schemes such as the continuous 3-generation cross between York (Y), Landrace (L), and Pietrain (P) hybrid sows and Pietrain boars. Semen samples were collected from 11 sexually mature boars from two sire lines. Samples were analysed using the ISAS^®v1 system to evaluate eight kinematic variables of sperm velocity, progressiveness and undulations. Four morphometric parameters of sperm head size (length, width, area and perimeter) were analysed. Bayesian analysis revealed relevant differences in four kinematic variables (VSL, LIN, STR and WOB) between sire lines, with a probability of relevance (P_R) of 0.79–0.91, and Pietrain boars were associated with higher progressive motility compared with Duroc x Pietrain boars. Moreover, there were relevant differences in all morphometric variables (P_R = 0.82–0.85) between sire lines. The dam line Y-L-50 (½ Y × ½ L) had higher total born per litter and piglets born alive, and YLP-75 (¹/₈ Y × ¹/₈ L × ³/₄ P) was associated with higher values of litter weight at birth (highest posterior density region at 95% = 9.92, 16.41 kg). There are relevant differences in kinematic variables between the assessed sire lines and the differences in morphometric and litter size variables were also relevant. The York-Landrace hybrid sows had higher total born per litter and piglets born alive, and there were relevant differences when compared with YLP-50 (¼ York × ¼ Landrace × ½ Pietrain). Differences in kinematic and morphometric variables between sire and dam lines related to fertility need to be further studied.     

Heritability and genetic correlation estimates of semen production traits with litter traits and pork production traits in purebred Duroc pigs

We estimated heritabilities of semen production traits and their genetic correlations with litter traits and pork production traits in purebred Duroc pigs. Semen production traits were semen volume, sperm concentration, proportion of morphologically normal sperms, total number of sperm, and total number of morphologically normal sperm. Litter traits at farrowing were total number born, number born alive, number stillborn, total litter weight at birth, mean litter weight at birth, and piglet survival rate at birth. Litter traits at weaning were litter size at weaning, total litter weight at weaning, mean litter weight at weaning, and piglet survival rate from birth to weaning. Pork production traits were average daily gain, backfat thickness, and loin muscle area. We analyzed 45,913 semen collection records of 896 boars, 6,950 farrowing performance records of 1,400 sows, 2,237 weaning performance records of 586 sows, and individual growth performance records of 9,550 animals measured at approximately 5 mo of age. Heritabilities were estimated using a single-trait animal model. Genetic correlations were estimated using a 2-trait animal model. Estimated heritabilities of semen production traits ranged from 0.20 for sperm concentration to 0.29 for semen volume and were equal to or higher than those of litter traits, ranging from 0.06 for number stillborn and piglet survival rate at birth to 0.25 for mean litter weight at birth, but lower than those of pork production traits, ranging from 0.50 for average daily gain to 0.63 for backfat thickness. In many cases, the absolute values of estimated genetic correlations between semen production traits and other traits were smaller than 0.3. These estimated genetic parameters provide useful information for establishing a comprehensive pig breeding scheme.     

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.     

“In vitro” capacitation and further progesterone‐induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa

The aim of this study was to determine if the achievement of the “in vitro” capacitation (IVC) status and subsequent progesterone‐induced “in vitro” acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono‐ and bi‐dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono‐dimensional Western blot in non‐capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi‐dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non‐capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.     

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。     

牛孕激素受体SNP分析及其与精液品质的关系

孕激素受体(PR)对于正常雌性动物生殖功能的实现具有重要调节作用,该基因在雄性动物中生物学作用尚不十分清楚。本研究通过PCR-SSCP方法对种公牛群体的PR基因所有外显子及5′UTR多态性进行检测。结果表明:PR基因外显子4、外显子8及5′UTR均检测到了多态性位点,其他外显子没有发现遗传多态性;测序表明外显子4发生了A20G突变,没有导致氨基酸的改变,外显子8发生G86T和A207T突变,其中G86T突变导致所编码的G(甘氨酸)变成V(缬氨酸),A207T突变导致所编码的Q(谷氨酰胺)变成H(组氨酸),5′UTR处存在G-1809A和C-1808G碱基突变。最后用最小二乘分析方法对其基因型和生产性状进行了相关性分析,第4及第8外显子的遗传多态性与部分精液品质性状呈显著相关。     

Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain.

2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2 × 10⁹ cells/ml in pre-freezing extender; equilibration of diluted semen at 5°C, 20 vs 40 min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5°C, 1 vs 30 min; thawing at 60°C for 10 vs 50°C for 30 sec. Spermatozoa viability (EtBr exclusion procedure – stress test), mobility (Accudenz® swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen.

3. The lower SWC (1.5 × 10⁹ cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively.

4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol.

5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed.

6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa.