微量元素锌、铜对种公牛精液品质的影响

在对种公牛精液质量和微量元素营养状况分析的基础上,为了研究锌、铜在改善全群种公牛精液品质,治疗种公牛常年精液品质不良、性欲减退的效果,选择成年种公牛进行补充锌、铜的饲养试验。对试验前后精液品质及血液、精液、牛毛中微量元素含量进行了测定,现将试验结果报告如下。     

Genomewide analysis of bull sperm quality and fertility traits

Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein–Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high‐density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD‐adjusted Bonferroni <0.05) with the non‐compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non‐compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.     

Effect of pH on rheotaxis of bull sperm using microfluidics

The aim of the present research is to study the effect of pH values on the sperm rheotaxis properties. Semen collected from bulls was diluted with SOF medium (1:10). pH of the medium was adjusted using a digital pH meter to the following pH values: 6.0, 6.2, 6.4, 6.4, 6.8, 7.0. All kinetic parameters of sperm (n = 3,385) were determined through a computer‐assisted sperm analysis (CASA) system using microfluidic devices with controlled flow velocity. The following parameters were determined: total motility (TM%), positive rheotaxis (PR%), straightline velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, as VSL/VCL, %), beat cross‐frequency (BCF, Hz) and curvilinear velocity (VCL, μm/s). Nitric oxide, calcium and potassium were estimated in semen at different pH values. To confirm the effect of nitric oxide and K⁺, we used sodium nitroprusside (an NO donor) and KCL as (a K⁺ donor) to see their effect on sperm PR%. The results showed no difference in TM% at pH (6–7). The PR% was the lowest at pH 6 and 7. The best parameters for the PR% were at pH 6.4–6.6. The concentration of Ca⁺² did not change at different pH values. The mean NO values decreased with the increase of pH; however, the mean values of K⁺ increased with the increase of pH. Addition of high concentration of NO and K⁺ to the semen media at fixed pH level had a negative effect on TM% and PR%. In conclusion, the bull sperm had the best rheotaxis properties at pH 6.4–6.6 and sensitive to the change of seminal NO and K⁺.     

Effect of freezing bull semen in two non‐egg yolk extenders on post‐thaw sperm quality

Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post‐thaw sperm quality was evaluated by computer‐assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56‐day non‐return rates were evaluated. Semen frozen in the liposome‐based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin‐based extender. Chromatin integrity and production of live H₂O₂+ reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome‐based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56‐day non‐return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66% ± 0.8 and 65% ± 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome‐based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.     

提高种公牛精液利用率的试验研究

利用冷冻精液进行人工授精的技术在我省应用已有近三十年的历史,在推动全省牛品种改良发展方面起到了举足轻重的作用。饲养较少的种公牛生产出较多的优质冻精,即提高种公牛精液的利用率,是每个种公牛站长期以来一直追求的目标。种公牛精液冷冻效果的好坏是影响种公牛精液利用率的重要因素之一,而种公牛精液冷冻效果除受环境、气候等因素影响外,还受种公牛品种、年龄、个体等因素的影响。通过试验找出种公牛不同品种、不同年龄及不同个体对精液冷冻效果的影响,从而科学的利用种公牛精液,为有效提高种公牛精液利用率提供依据。     

种公牛微量元素营养状况对精液品质的影响

为了查找种公牛精液品质下降的原因并探讨精液品质与微量元素的关系,试验测定了种公牛基础日粮及血液、被毛、精液中锌(Zn)、铜(Cu)、铁(Fe)、锰(Mn)、铬(Cr)5种微量元素含量。结果表明:饲料中Zn、Cu含量明显低于美国NRC推荐标准,Fe、Mn、Cr三种元素能满足种公牛的营养需要;精液品质正常牛及异常牛血液、被毛、精液中Fe、Mn、Cr差异不显著(P>0.05),被毛、血液中Zn、Cu两种元素含量差异显著(P<0.05),精液中Zn元素含量差异极显著(P<0.01)、Cu元素含量差异显著(P<0.05)。     

Detection of mycoplasmas in experimentally-infected bull sperm

Bull sperm pellets experimentally infected with various concentrations of Mycoplasma (M.) bovis, M. bovigenitalium, and M. californicum were stored several months at -196 degrees C and were subsequently tested for mycoplasmas, using different methods and culturing media. M. bovis was reliably identified by laboratory diagnosis up to a concentration of 10(2) cfu/pellet. M. bovigenitalium and M. californicum were found to make higher demands on the quality of culturing substrates. Recommended are 1-h broth incubation and 5-d broth breeding.     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Detection certainty of the contamination of bull sperm with mycoplasma

Tests for presence of mycoplasmas were conducted on 20 insemination bulls known as mycoplasma spreaders, with 5 sperm pellet batches of 20 pellets each being investigated for each animal. Mycoplasma contamination was positively recorded from 83 of the above 100 batches. Mycoplasma (M.) bovigenitalium, M. californicum. M. arginini, M. bovirhinis, and Acholeplasma laidlawii were typed by means of indirect immunofluorescent technique, which confirmed the presence also in the GDR of mycoplasma species described in the literature and detected in sperm samples. The solid culturing media used in the above tests, medium-B agar and cattle blood agar with staphylococcal nutrix, proved to be equally suitable for isolation of mycoplasma from sperm samples. Mycoplasma was positively identified in about one third of all pellets/batches tested. 3 pellets to one batch should be sufficient a random sample size from which to obtain information at least very close to real contamination.     

Validating the assessment of bull sperm morphology by veterinary practitioners

The objective of this study was to validate the assessment of bull sperm morphology done by veterinary practitioners. Out of 1606 bulls, 1400 (87.2%) and 1344 (83.7%) were designated by practitioners and an experienced andrologist, respectively, as having > 70% morphologically normal sperm. In 92% of the evaluations, there was agreement between the designations chosen.     

The ultrastructure of bull sperm membranes prepared by freeze etching

Intramembraneous particles represent protein components of the membrane. It is assumed that their number and distribution are in correlation with the level of the metabolic activity of membranes. In the cytoplasmic membrane in the postacrosomal region (PF) near the posterior ring the particles are arranged in form of palisade rows. In the remaining membranes of the head, irregularly distributed particles were observed. Their number at individual places varied. Their size was not equal either. The areas without particles were also found out. The particles in the cytoplasmic membrane of the flagellum (PF) formed clusters and they had circumferential orientation in the direction of mitochondria. At the place of the annulus the number of particles increased. The distribution of particles was irregular in the cytoplasmic membrane of the main section.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

NYD-SP27基因对绵羊精子质量及精子中cAMP信号通路的影响

为了研究睾丸发育特异性蛋白-27基因(testis development specific protein gene 27, NYD-SP27)基因对绵羊精子活率、畸形率及精子内cAMP信号通路中酶类表达量影响,分别将NYD-SP27基因的过表达载体、干扰载体质粒注射到绵羊的睾丸中,通过电刺激采精法采集绵羊精液,对绵羊精子进行染色,检测其活率、畸形率,并通过ELISA方法检测精子中cAMP信号通路中相关酶类的表达量。结果显示:与对照组相比,过表达组精子活率变化不明显,而干扰组有较明显的升高,精子畸形率差异不明显;与过表达组、干扰组绵羊精子中环磷酸腺苷酸的表达量差异极显著(P<0.01);与过表达组绵羊精子中PKA的表达量差异不显著(P>0.05),而干扰组显著升高(P<0.05)。综上所述,干扰NYD-SP27基因对精子中环磷酸腺苷酸和PKA的表达具有调控作用,并对绵羊精子活率具有一定的提高效果。本研究为进一步研究NYD-SP27基因调控绵羊精子发生及获能的分子机制奠定基础。     

Effect of various levels of dissolved oxygen on reactive oxygen species and cryocapacitation‐like changes in bull sperm

The present investigation was carried out to study the effect of various levels of dissolved oxygen (DO) on reactive oxygen species (ROS) and cryocapacitation‐like changes in bull sperm. Egg yolk–Tris–glycerol (EYTG) extender was split into four subextenders; viz., Extender I (control; no flushing with liquid nitrogen (LN₂)), Extender II, Extender III and Extender IV were flushed with LN₂ for 40, 16 and 8 min, respectively. The DO levels were standardized to 11.7, 2, 4 and 8 ppm, respectively, in control (Extender I), Extender II, Extender III and Extender IV. Ejaculates with mass motility of ≥ 3+ were divided into group I (diluted with Extender I), group II (diluted with Extender II), group III (diluted with Extender III) and group IV (diluted with Extender IV) up to 80 × 10⁶ sperm/ml. Extended semen samples were packed in French mini straws (0.25 ml), equilibrated and cryopreserved. Semen samples were evaluated at prefreeze and post‐thaw stage for various parameters (DO, progressive motility (PM), viability (VIB), acrosomal integrity (AI), hypo‐osmotic swelling (HOS) test, ROS, cholesterol (C) and phospholipid (P). The percentage of PM, VIB, AI, HOS test, cholesterol (C) and phospholipid (P) levels, and capacitated sperm were significantly (p < 0.05) higher in groups III and IV as compared to groups I and II. However, the acrosome‐reacted sperm (%; pattern AR) were significantly (p < 0.05) decreased in group III as compared to all other groups. Besides the proportion of sperm displaying tyrosine‐phosphorylated pattern, EA (fluorescence at both equatorial and anterior acrosomal regions, i.e. high capacitation level) was significantly (p < 0.05) reduced in group III compared to all other groups. In conclusion, varying DO levels in the extender significantly affect sperm quality, ROS production and capacitation‐like changes in bulls.