Stallion Sperm Selection: Past,Present, and Future Trends

Although several selection techniques are available for processing spermatozoa, only colloid centrifugation has been used to any extent in this field, starting with density gradient centrifugation and progressing more recently to single-layer centrifugation (SLC). SLC through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility and normal morphology from stallion semen. The method is easier to use and less time-consuming than density gradient centrifugation, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The potential applications of SLC in equine breeding are as follows: to improve sperm quality in artificial insemination doses for “problem” ejaculates, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.     

The Effect of Activation of Granulocytes on Enzyme Release and Hydrogen Peroxide and Superoxide Production in Buffaloes

Singh, V.K., More, T. and Singh, S., 1997. The effect of activation of granulocytes on enzyme release and hydrogen peroxide and superoxide production in buffaloes. Veterinary Research Communications, 21 (4), 241-247Polymorphonuclear cells kill microorganisms by the stock of antibiotic proteins and peptides stored in their lysosomal granules and have the ability to produce reactive oxygen intermediates (ROI) such as H₂O₂, O ₂ ^– , and HOCl. Since the components involved in the microbicidal functions of buffalo (Bos bubalis) polymorphonuclear cells (PMN) have not been characterized, an assessment was made of the levels of various enzymes, the extent of extracellular release of these enzymes, and also their ability to produce H₂O₂/O ₂ ^– upon activation with opsonized zymosan (OZ) or lipopolysaccharide (LPS). Using GPC-HPLC, OZ was shown to be a more potent secretagogue than LPS, causing a significantly greater release of low-molecular-weight components. Varying levels of the enzymes (myeloperoxidase, lactate dehydrogenase, acid and alkaline phosphatases, -galactosidase, -D-glucuronidase, elastase and lysozyme) were recorded in the buffalo PMN and both the activators (OZ and LPS) caused significant release of all the enzymes except alkaline phosphatase. Both the activators also caused a significant increase in H₂O₂/O ₂ ^– production by the PMN. However, OZ caused a more pronounced activation than LPS. The studies revealed the presence of oxygen-dependent and oxygen-independent microbicidal systems with buffalo PMN, which responded more effectively to zymosan activation.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Colloidal Centrifugation of Stallion Semen: Changes in Sperm Motility,Velocity, and Chromatin Integrity during Storage

The current study investigated the changes in sperm quality (motility, velocity, and chromatin integrity) occurring during storage at room temperature or 5°C for up to 48 hours in spermatozoa after extension or single-layer centrifugation (SLC) through Androcoll-E. In unselected samples, all parameters of sperm quality deteriorated significantly during storage (P < .01), although the deterioration was faster at room temperature (22–30°C) than for cool storage (P < .01). The SLC-selected spermatozoa had higher motility, velocity, and chromatin integrity than the overall unselected population (motility: selected 85 ± 10%, unselected 56 ± 13%; P < .001; velocity: selected 85.1 ± 13 μm/second, unselected 63.5 ± 15 μm/second; P < .001; and DFI selected 12.2 ± 4.8 μm/second, unselected 23.6 ± 7.4 μm/second; P < .001). Furthermore, sperm quality did not deteriorate with storage in the SLC-selected samples, either at room temperature (22–30°C for 24 hours) or cooled to 4°C (for at least 48 hours), whereas a significant deterioration in sperm quality was observed in the unselected sperm samples (P < .01). Thus, room temperature storage of SLC-selected spermatozoa may be an option for insemination doses from stallions whose spermatozoa do not tolerate cooling. In addition, a new sperm analyzer, the Qualisperm, showed good correlation with subjective motility assessment (r = 0.8, P < .001), was user-friendly, and provided a reasonable volume of data. This instrument may be a useful adjunct to sperm quality assessment at the stud.     

不同催青处理的二化性家蚕体内过氧化氢代谢

研究了25℃明催青和15℃暗催青对二化性家蚕品种丰一体内的H2O2代谢影响。在催青期,前一种处理的蚕卵体内H2O2代谢水平在中后期呈U型变化,过氧化氢酶(CAT)活性在后期迅速上升;后一种处理的蚕卵体内H2O2代谢水平在第16天出现峰值,CAT活性在后期缓慢上升。在幼虫期,两种催青处理的H2O2代谢基本相似,均是H2O2代谢水平随龄期增加而逐步下降,CAT活性在1~4龄逐步下降,而在5龄期有所回升。在蛹期,前一种处理的H2O2代谢水平分别在第2天和第9天出现峰值,CAT活性分别在第3天和第6天出现峰值;后一种处理的H2O2代谢水平在第4天出现峰值,CAT活性在第5天出现峰值。上述结果表明,不同催青条件显著改变了家蚕催青期和蛹期的H2O2代谢,滞育诱导阶段的家蚕H2O2代谢可能与滞育激素(DH)的表达密切相关。     

The Effect of Brucella abortus on Hydrogen Peroxide and Nitric Oxide Production by Bovine Polymorphonuclear Cells

The effect of Brucella on the generation of microbicidal reactive oxygen and nitrogen metabolites by bovine peripheral polymorphonuclear cells (PMNs) was investigated. The PMNs were recovered from the peripheral blood of control calves and experimental calves previously vaccinated against brucellosis. Significantly larger quantities of NO and H₂O₂ were generated by PMNs from control and experimental calves following activation by heat-killed whole cells or outer membrane protein of Brucella abortus than by non-activated cells (p<0.05–0.01). In contrast, generation of H₂O₂ and NO decreased when PMNs were exposed to the lipopolysaccharide of Brucella. However, the generation of H₂O₂ and NO by activated PMNs from the control and experimental calves did not differ significantly.     

Comparison of the Effects of Four Freezing Methods on Motility Characteristics,Morphology, and Viability of Postthaw Stallion Epididymal Sperm

Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freeze-thaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period.     

冷藏期家蚕滞育卵和即时浸酸卵的H2O2含量及过氧化氢酶基因表达的变化

将产下后48h的家蚕滞育卵和即时浸酸卵进行低温(5℃)冷藏处理30d以上,滞育卵孵化率显著上升,而即时浸酸卵孵化率显著下降。为了探讨低温处理对滞育卵和即时浸酸卵的孵化产生不同影响的分子机制,测定了冷藏期间2种卵中的H2O2含量、过氧化氢酶(CAT)基因mRNA转录水平和CAT活性变化。结果表明:2种卵在冷藏期间随着卵中的H2O2含量显著增加,CAT基因mRNA转录水平也上调;虽然冷藏后的10～70d,2种卵中的H2O2含量、CAT基因mRNA转录水平及CAT活性都显著上升,但滞育卵的H2O2含量仍显著高于即时浸酸卵,而CAT基因mRNA转录水平和CAT活性则显著低于即时浸酸卵。即时浸酸卵在冷藏过程中H2O2含量显著增加,可能造成了对胚胎的氧化伤害,从而降低其孵化率。然而,滞育卵在冷藏过程中的CAT基因mRNA转录水平及CAT活性相对较低,H2O2相对较高,胚胎却未受到氧化伤害,推测低温处理可能诱导了滞育卵中其它抗氧化反应。     

蓖麻蚕滞育与过氧化氢代谢的关系

为了探讨蓖麻蚕滞育的机理,研究了蓖麻蚕蛹H2O2代谢的变化,并比较了在5℃冷藏滞育条件下的差异。结果表明,常温环境蓖麻蚕蛹期的H2O2含量呈减少趋势,第3~5天急速变化,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性在第5天出现活性峰,故蛹期第3~5天是蓖麻蚕生理变化的一个敏感时期。第3天开始蛹滞育条件5℃冷藏,雄蛹在30 d前后、雌蛹在80 d左右H2O2含量出现快速改变;SOD和CAT活性一直呈下降趋势,但自30 d开始CAT活性出现快速下降过程,5℃冷藏30 d是蓖麻蚕蛹H2O2代谢的临界期生理时间,这与蚕蛹的30 d冷藏阈值时间吻合。     

3-氨基三唑与过氧化氢共同处理对家蚕滞育发动的影响

研究有效阻止家蚕滞育发动的措施发现 :在家蚕滞育发动期间 ,仅用过氧化氢酶 (catalase ,CAT)的专一性抑制剂 3 氨基三唑 (3 amino 1,2 ,4 triazole ,AT)处理滞育性蚕卵仍不能阻止家蚕滞育发动 ;但先用AT处理抑制CAT活性后 ,再用H2 O2 处理可有效阻止家蚕滞育发动。     

Hydrogen Peroxide Production by Mycoplasma bovis and Mycoplasma agalactiae and Effect of In Vitro Passage on a Mycoplasma bovis Strain Producing High Levels of H2O2

Hydrogen peroxide (H2O2) production and oxygen uptake during the oxidation of NADH and L-alpha-glycerophosphate (GP) by lysed cells was determined for the type and field strains of Mycoplasma bovis and M. agalactiae. NADH oxidation by all the strains showed variable production of H2O2 ranging from 0 to 1.21 mol/mol O2 taken up. All strains were unable to oxidize GP, showing absence of GP oxidase activity. Some strains were identified that produced relatively high levels of H2O2 (> 1.0 mol/ mol O2 taken up). In vitro passage of M. bovis strain 119B96 showed reduced H2O2 production: 0.52, 0.16, and 0.07 mol/mol O2 taken up after the 50th, 100th and 200th passages, respectively. SDS-PAGE analysis showed the loss of a protein band of 32 kDa after 50 passages. These preliminary studies show that not only does H2O2 production by potentially pathogenic Mycoplasma spp. vary in the field but also that similar alterations can be induced by passage in culture. In the latter case, at least in one M. bovis strain, this alteration has been shown by SDS-PAGE to be associated with a loss of specific protein production. Further study of these phenomena is essential background for the production of more efficient vaccines for mycoplasmas.     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

Donkey Feeding During Maintenance,Pregnancy, and Lactation: Effects on Body Weight,Milk Production,and Foal Growth

We evaluated the daily intake in donkeys during maintenance, late pregnancy, and early lactation. The growth curves of the foals in the first eight weeks of life and the milk production in lactating jennies were also investigated. Donkeys were separated into two groups: seven pregnant jennies (group 1: pregnant/lactating) and seven nonpregnant, nonlactating jennies (group 2). The groups were fed two different diets each. The feeding period for group 1 covered eight weeks before parturition and eight weeks postpartum. Group 2 was managed during the same time period (16 weeks). Diet 1 consisted of ad libitum hay, which was fed to group 1 during late pregnancy and to group 2 from weeks 1–8. Diet 2 consisted of the same ad libitum hay supplemented with 2 kg/head/day of concentrate, which was fed to group 1 during lactation and to group 2 from weeks 9–16. The daily dry matter intake (diet 1) was 2.56 kg/100 kg of body weight (BW) for nonpregnant jennies and 31% lower in the pregnant donkeys. In the lactating jennies, the total dry matter intake was similar to the nonpregnant group fed diet 2. The nonpregnant jennies lost 1.9% of their initial BW when fed diet 1, whereas they gained 8.4% of their initial BW when fed with diet 2. A weight loss was also found in the lactating donkeys. The foals more than doubled in their birth weight within two months.     

大黄和大黄素对体外瘤胃发酵甲烷、氢气和挥发性脂肪酸生成的影响

本试验研究不同添加量的大黄和大黄素对体外瘤胃发酵甲烷、氢气和挥发性脂肪酸生成的影响。选用3只装有永久性瘤胃瘘管的成年浏阳黑山羊作为瘤胃液供体,设对照组(A,没有任何处理)、大黄组(B1、B2、B3和B4组,添加量分别为0.5、1.0、2.0和2.5 mg/mL)和大黄素组(C1、C2和C3组,添加量分别为0.06、0.12和0.24 mg/mL),进行24 h体外模拟瘤胃发酵试验。结果表明:1)随着添加量的增加,大黄组产气量、起始底物降解速率均呈先升高后降低的二次曲线变化趋势(P0.05),大黄素组均呈线性下降的变化趋势(P0.05);2)与对照组相比,添加大黄(≥1.0 mg/mL)和大黄素显著降低了甲烷产量,并随其添加量的增加呈线性下降的变化趋势(P0.05);3)与对照组相比,添加大黄(≥2.0 mg/mL)和大黄素(≥0.24 mg/mL)后,氢气含量(发酵瓶顶部空间)及产量显著增加(P0.05),并随其添加量的增加呈线性上升的变化趋势(P0.05);4)与对照组相比,添加大黄(≥1.0 mg/mL)和大黄素显著降低了乙丙比,并随其添加量的增加呈线性下降的变化趋势(P0.05)。结果提示,适当添加大黄(≤1.0 mg/mL)没有改变起始底物降解速率,可能不会影响反刍家畜对饲料的降解、消化和利用;而大黄素会显著降低起始底物降解速率,影响反刍家畜对饲料饲草的降解、消化和利用。添加大黄(≥1.0 mg/mL)和大黄素,可以降低甲烷的生成,增加氢气产量,并且改变瘤胃的发酵模式,使其向丙酸发酵型转变。     

Chemical Activation with a Combination of Ionomycin and Dehydroleucodine for Production of Parthenogenetic,ICSI and Cloned Bovine Embryos

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μm Io for 4 min followed by 5 μm DhL concentration and after 3‐h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6‐dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI‐embryos or SCNT‐embryos is reported here for first time.     

The Effect of Herbicide Application During the Winter on Forage Production,Animal Performance,and Grain Yield of Winter Wheat

Winter wheat (Triticum aestivum L.) is used for both forage and grain production throughout the U.S. Southern Great Plains region. Management practices to increase grain yield may affect winter forage production and as a result, animal performance. The objective of this study was to determine the effect of applying herbicides in the winter to control broad leaf weeds on winter and spring forage production, animal performance, and subsequent grain yield. Four 8-ha wheat pastures at each of two locations were established under no-till practices each fall, then randomly assigned within location to receive one of the following four treatments: 1) no winter herbicides, 2) chlorsulfuron, 3) chlorsulfuron plus metsulfuron, or 4) triasulfuron. Herbicides were applied in December (yr 1) or January (yr 2) combined with 2,4-dichlorophenoxyacetic acid (2,4-D). Stocker steers were used as grazers to determine animal performance during winter (December to March) and spring (March to May) grazing periods. Animal performance and beef production per ha were not (P>0.10) affected by the application of herbicides. Animal productivity was related to climatic conditions, which affected both the energy required by the animal for maintenance and the amount of forage produced. Winter and spring forage production was not affected (P>0.10) by herbicide application. The amount of nonwheat biomass present at grain harvest was lower (P<0.03), the harvest index was higher (P<0.01), and the number of seed head was higher (P<0.01) for herbicide-treated plots than for control plots. Among the herbicides used, triasulfuron increased the number of seed heads and grain yield. Applying herbicides to winter wheat in the winter to control broadleaf weeds did not affect forage or animal production but did increase harvest index.     

Reproductive Development of Santa Inês Rams During the First Year of Life: Body and Testis Growth,Testosterone Concentrations,Sperm Parameters,Age at Puberty and Seminal Plasma Proteins

We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two‐dimensional SDS‐PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 ± 0.7 to 54.3 ± 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non‐significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 ± 0.05 to 1.14 ± 0.24 × 10⁹ cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 ± 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 ± 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.