Analysis of the motility of bull sperm in fresh ejaculates using computer technology]

The activity of spermatozoa was measured in the fresh non-diluted ejaculates of 10 breeding bulls, using the HTM motility analyzer version 7. The average path speed was 83.6 microns.s-1, the average progressive speed was 48.2 microns.s-1 and the average straightness of the movement path was 58%. The spermatozoa were classified and it was found that most frequently they moved at a path speed of 60-80 microns.s-1 (28.2%) and at a progressive speed of 20-40 microns.s-1 (33.2%). The path straightness classes above 40% included evenly distributed numbers of spermatozoa; in classes with a less than 40% straightness the numbers of spermatozoa were much smaller. These data are characteristic of fresh undiluted bull ejaculates, suitable for artificial insemination.     

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates

Background: Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation,was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars,but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.Results: Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.Conclusions: These data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.     

The effect of cooling to different subzero temperatures on dog sperm cryosurvival

The objective was to assess the effect of cooling to different subzero temperatures around ice formation (?5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris‐egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris‐egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 10⁶ cells/ml). Sperm were packaged in 0.5‐ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to ?3, ?5 or ?7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to ?3 or ?5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was ?14.3 ± 2.05°C (mean ± SD); cooling to +5, ?3, ?5 and ?7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, ?3, and ?5°C produced no differences on sperm survival and plasma membrane fluidity after freeze–thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.     

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.     

The acrosin system of bull, boar and ram sperm]

Described in this paper is a technique by which to separate the components of the sperma acrosin system. Included in the method are extraction of all components by means of acetic acid, separation of acrosin inhibitors on Sephadex G 100 as well as biochemical determination of proacrosin and acrosin. While species-related peculiarities were of minor importance, alterations were found to occur to the acrosin system in response to deep-freeze preservation of bull, boar, and ram sperma. Those alterations grew manifest primarily through decline in total acrosin activity and shifting of the proacrosin-acrosin ratio in the direction of proacrosin activation. Detachability of membrane-bound acrosin inhibitors was increased with significance, following in-vitro capacitation of bull sperma under heparin action.     

Assessment of the morphology of frozen-thawed bull sperm in relation to its cryopreservation for artificial insemination

The morphology of sperm in raw semen was compared with that of the live sperm in semen which had been frozen and thawed. The thawed semen was stained with 6-carboxyfluorescein diacetate and propidium iodide and examined by fluorescence microscopy; smears of the raw semen were stained with eosin and nigrosin. Thirty-four ejaculates from 24 bulls of various breeds were examined. There were fewer abnormal heads, detached heads, coiled tails and proximal cytoplasmic droplets/pseudodroplets in the thawed semen than in the raw semen, there was no change in the number of bent tails, but the number of distal cytoplasmic droplets/pseudodroplets increased. There were no significant differences in morphology between ejaculates which passed or failed the osmotic resistance test after thawing, but failed batches tended to have more distal cytoplasmic droplets/pseudodroplets.     

使用犊牛鲜奶替代品有助于生产性能改善

使用犊牛奶粉代替鲜奶饲喂犊牛，生长性能更好，这就是全球的许多农场主正在用犊牛奶粉代替鲜奶饲喂犊牛的原因。为了检验犊牛奶粉在中国使用的效果，我们分别在北京、四川、广州安排了４家奶牛场用喜利康犊牛奶粉饲喂犊牛的试验。１试验结果１．１北京市南口农场一分场　见表１。１．２广州市国营新洲奶牛场见表２。１．３ 四川省沙河堡乳牛场见表３。１．４广州市云燕畜牧发展公司　见表４２结论与讨论２．１喜利康犊牛奶粉可以完全替代除初乳以外的全部母乳饲喂犊牛，其增重效果要优于母乳。从表１、表２、表３可以看出，喜利康组犊牛的…     

Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl^®, 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh – 0 hr, pre-freeze – 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 10⁹ sperm/ml vs. 1.37 ± 0.10 × 10⁹ sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.     

The detection of in vitro capacitation of bull sperm by heparin treatment

The capacitating effect of heparin upon spermatozoa from original and deep-frozen semen was characterised, using new methods for detection of inducible acrosomal reaction and heparin-mediated sperm aggregation, and was compared with frequently used capacitation by media of high ion strength. Heparin treatment was undertaken also by means of two culture media, "defined medium" (DM) and TCM 199, with 10% fetal calf serum. Higher motility was maintained by means of 10 I.U. of heparin/ml (= 77 micrograms/ml) which also proved helpful in achieving higher capability of inducible acrosomal reaction, as compared to pretreatment, using media of high ion strength. This applied to both fresh and deep-frozen sperm. The highest level of inducible acrosomal reaction was achieved after 2 hours of heparin action on fresh sperm and 30 minutes of action on deep-frozen sperm. That highest value was at its maximum, when TCM with 10% fetal calf serum had been used. This was the medium, after all, in which photometrically recorded aggregation of motile spermatozoa was at its fastest rate, reaching its maximum after about 60 minutes. The photometrically recorded activated motility of spermatozoa occurred more frequently in TCM, as compared to DM. Preparation of bull sperm in TCM 199 with fetal calf serum and heparin may be recommended as an effective and time-saving method for in vitro capacitation.     

荷斯坦公牛PRNP多态性与精子活力相关性及抗病性评估

为分析荷斯坦公牛朊蛋白基因(PRNP)多态性与精子活力的关系,并对其抗病性进行评估,选育抗病公牛,本实验以公牛精液为样品,研究脚基因中12bp、23 bp和24 bp 3个片段的插入/缺失多样性、基因mRNA转录水平及其与精子活力等指标的关系.结果表明,12bp和23 bp在群体中均得到3种基因型,24 bp只有2种基因型.不同基因型群体的mRNA转录水平存在显著差异,而基因型与精液产量和活力等指标存在相关性,抗病力和精子活力指标存在负相关.本研究中3个片段的插入-缺失多样性可以作为公牛选育的辅助标记.     

Timing of magnesium supplementation administered through drinking water to improve fresh and stored pork quality

Thirty-two pigs were used to determine the timing effect of magnesium (Mg) supplementation given through drinking water on pork quality. Pigs (16 barrows and 16 gilts) were individually penned, provided 2.7 kg of feed (0.12% Mg) daily (as-fed basis), and allowed free access to water via a nipple waterer for the duration of the study. After 5 d of adjustment, pigs (120 +/- 0.8 kg BW) were allotted randomly by weight and sex to 900 mg/L of supplemental Mg from magnesium sulfate heptahydrate in drinking water for -6, -4, -2, or 0 d relative to slaughter. The LM and semimembranosus (SM) muscles were removed 24 h postmortem. Retail display storage was simulated for 8 d, and the LM was vacuum-packaged for 25 or 50 d at 4 degrees C. Magnesium did not affect the pH of the LM at either 45 min (P = 0.15) or 24 h postmortem (P = 0.23). However, the pH of the SM at 24 h postmortem tended to be greater (P = 0.08) for pigs consuming Mg for 2 d than for those not supplemented. Fluid loss after 8 d of storage was less (P < 0.05) in the LM of pigs supplemented with Mg for 6 d than in those without supplementation. Furthermore, fluid loss from the SM of pigs provided supplemental Mg for 2 d, but not for 4 or 6 d, was lower (P < 0.05) on each day of retail display than the SM of unsupplemented pigs. Minolta L*, a*, and b* color measurements of the LM during display storage were not (P > 0.10) affected by Mg supplementation. However, Mg supplementation for 2 or 4 d decreased paleness (lower L* value) after 25 d (P < 0.05), but not 50 d (P > 0.10) of vacuum-packaged storage. Magnesium addition for 2 d decreased the extent of oxidation (thiobarbituric acid-reactive substances) of the LM after 4 d of display storage compared with 0 d of Mg (P < 0.05). Oxidation of the SM during 8 d of display storage increased linearly (P < 0.05) as duration of supplementation increased from 2 to 6 d but did not differ (P = 0.22) from 0 d of Mg supplementation. Although the response to Mg supplementation was variable, supplementation for 2 d before slaughter was considered most efficacious because of the following: decreased fluid loss from the SM, and lower lipid oxidation formation in the LM during retail storage; a darker, more desirable LM color after 25 d of vacuum-packaged storage; and cost reductions compared with longer durations.     

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.     

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.