Effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa

The effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris–egg yolk–glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50‐ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post‐freeze‐thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris–egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm.     

Serological evidence of Neospora caninum infection in mithun (Bos frontalis) from India

Studies conducted on mithuns maintained at National Research Centre on mithun, Indian Council of Agricultural Research (ICAR), Nagaland, India and mithuns found in free-ranging condition of Nagaland revealed that the overall prevalence of antibodies to Neospora caninum in mithun was 10% (95% CI=5-15) when detected by a commercially available competitive enzyme-linked immunosorbent assay test. Highest (prevalence rate=16, 95% CI=8-24) seroprevalence was found in mithuns above 3 years of age and lowest (prevalence rate=2, 95% CI=0-6) in mithuns of 2-12 months old. No statistically significant difference was observed between male (prevalence rate=7, 95% CI=0-14) and female (prevalence rate=12, 95% CI=6-18) seroprevalences. The seroprevalence was found to be higher (prevalence rate=20, 95% CI=9-31) in mithuns found in free-ranging condition in comparison to mithuns kept in semi-intensive system (prevalence rate=5, 95% CI=1-9). This is probably the first report on serological evidence of N. caninum infection in mithun. The possible role of sylvatic fauna in the epidemiology of N. caninum infection mithun is also discussed in brief.     

Rumen microbial variation and nutrient utilisation in mithun (Bos frontalis) under different feeding regimes

The aim of the study was to investigate the effect of feeding different diets on fermentation, enzyme activities and microbial population in the rumen fluid of mithun (Bos frontalis). In a randomized block design, 20 male mithun (6–8 months of age, 152 ± 12.6 kg body weight) were randomly divided into four experimental groups (n = 5/group) and fed experimental diets ad libitum for 180 days. The diet R₁ contained tree foliages (TF), R₂ comprised of 50% concentrate mixture (CM) and 50% TF, R₃ contained 50% CM and 50% rice straw, and R₄ contained 50% CM, 25% TF and 25% rice straw. Rumen liquor was collected at 0 and 180 days of the experiment for estimation of different ruminal parameters and a digestion trial was conducted at the end of the experiment. Rumen fluid was analysed for pH, ammonia nitrogen (NH₃‐N), total‐N, ruminal enzymes, short chain fatty acid (SCFA) and microbial profile. The relative quantification of ruminal microbes was carried out with real‐time PCR using bacteria as the house keeping gene. The dry matter intake, nutrients digestibility, body weight gain, NH₃‐N, total‐N, carboxymethyl cellulase, avicelase, xylanase, amylase, protease and molar proportion of butyrate were (p < 0.05) higher in mithun fed R₂, R₃ and R₄ compared to those fed R₁ diet. In contrast, increased (p < 0.05) ruminal pH, molar proportion of acetate and, acetate to propionate ratio was recorded in mithun fed only TF than those fed concentrate supplemented diets. Similarly, an increase (p < 0.05) in the population of Fibrobacter succinogenes, Ruminococcus flavefaciens and total bacteria were evident in mithun fed R₂, R₃ and R₄ compared to those fed R₁. Therefore, it is concluded that TF 25% and/or rice straw 25% along with CM 50% may be fed to the growing mithun for improved rumen ecology, nutrient utilization and thus better performance under stall fed system.     

Milk protein polymorphisms in cattle (Bos indicus), mithun (Bos frontalis) and yak (Bos grunniens) breeds and their hybrids indigenous to Bhutan

In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β‐lactoglobulin (A, B, E and M), five molecular types of α_S1‐casein (A, B, C, E and X) and three molecular types of k‐casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24–40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle.     

Comparative nutrient utilization,growth, and rumen enzyme profile of mithun (Bos frontalis) and Tho-tho cattle (Bos indicus) fed on tree-leaves-based ration

A study was conducted to compare the nutrient utilization, growth, and rumen enzyme profile of mithun (Bos frontalis) and Tho-tho cattle (Bos indicus) reared in the same feeding and managemental conditions. For the purpose, male mithun (n = 8) and male Tho-tho cattle (n = 8) of 1.5 years age, selected from the farm of National Research Centre on Mithun, Nagaland, India, were fed on mixed-tree-leaves-based ration as per the requirement of NRC (2001) for cattle for 12 months. Average daily gain (ADG), average dry matter intake (DMI), and feed conversion ratio (FCR) for all animals were recorded. A metabolic trial was conducted at 6 months of the experiment to assess the digestibility coefficient of different nutrients and nutritive value of ration. At 12 months of the experiment, rumen liquor was collected from all animals and analyzed for rumen enzyme profiles, viz., carboxymethylcellulase, xylanase, α-amylase, β-glucosidase, α-glucosidase, urease, and protease. It was found that ADG (507.8 g vs 392.8 g), DM intake (6.59 vs 5.85 kg/day) and DMI/W^0.75 (98.75 g vs 91.00 g/day), crude protein intake (780 vs 700 g/day), and total digestible nutrient intake (3.65 vs 3.32 kg/day) were higher (p < 0.05) in mithun than cattle. The nitrogen balance was higher and FCR was better (p < 0.05) in mithun compared with cattle. The digestibility coefficient of different nutrients was similar (p > 0.05) between the species. The microbial enzyme profiles of mithun and cattle were not different (p > 0.05). The better growth performance of mithun than cattle as found in the present study clearly indicates that the mithun has higher genetic potential for growth than Tho-tho cattle of north-eastern hilly region of India.     

Determination of effective dosage of GH-releasing factor for blood GH responses in mithun (Bos frontalis)

A study was undertaken to determine the effective dosage of GH-releasing hormone (GRF) required to produce blood GH response in mithun (Bos frontalis), a semi-wild ruminant species. For the purpose, 12 mithuns averaging 11.5 months of age and 146 kg body weight (BW) were randomly assigned to receive GRF (n = 12), administered at 0 (normal saline), 5, 10 and 20 mug per 100 kg BW. Blood samples were collected prior to and after GRF administration at -60, -45, -30, -15, -10, -5, 0 min and 5, 10, 15, 30 and thereafter, at 15-min interval up to 8 h post-GRF were assayed for plasma GH. For all the dosages, the pre-treatment GH concentrations and corresponding area under GH response curve (AUC) were similar (p > 0.05). The post-GRF plasma GH responses to different dosages of GRF viz. 5, 10 and 20 mug per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those recorded in normal saline-treated controls. The GH responses to 10 and 20 mug GRF per 100 kg BW and corresponding AUCs were higher (p < 0.05) than those registered in mithuns administered with 5 mug GRF per 100 kg BW. Interestingly, post-GRF concentration of plasma GH and AUCs were not different for 10 and 20 mug GRF per 100 kg BW dosages. In all animals treated with GRF, a peak of GH was registered within 10 to 20 min post-GRF. Following 5 mug GRF per 100 kg BW, GH concentrations were maintained at higher level for 90 min post-GRF and thereafter became similar to that of controls and it was 435 min for 10 and 20 mug GRF per 100 kg BW dosages. In conclusion, our results suggest that 10 mug GRF per 100 kg BW is the dosage, which can be used for augmentation of mithun production.     

Genetic characterisation of the mithun (Bos frontalis) and studies of spermatogenesis, blood groups and haemoglobins of its hybrids with Bos indicus

The mithun (Bos frontalis) and its hybrids with Bos indicus were studied to provide further cytogenetic information which might throw light on the mechanisms of the male hybrid infertility and facilitate the establishment of a stable crossbreed. It was shown that compared with conventional cattle the mithun has a homozygous, species specific 2/27 centric fusion which reduced the diploid chromosome number from 60 to 58. This provided further proof that Robertson translocation-type rearrangements have been the major source of interspecies karyotype differences in the evolution of the Bovidae. In the mithun there was also significant polymorphism between centromeres of non-homologous chromosomes and there was heteromorphism between several homologous chromosomes which could possibly serve as useful genetic markers for breeding programmes. In F1 hybrids spermatogenesis progressed to a relatively advanced stage, without going so far as to produce spermatozoa. In back crosses to B indicus spermatogenesis progressed further so that spermatozoa could be seen, though not as numerous as in normal bulls. In most hybrids there were haemoglobin bands which corresponded either to Hb A or Hb B of cattle but were much wider. It was shown that these were a combination of Hb Mi derived from the mithun and Hb A or Hb B derived from B indicus. In a few hybrids there were only Hb Mi. In these cases Hb Mi had been present in both parents and proved that the dam was not a pure siri. The possible mechanism of hybrid male infertility is discussed including faults in the epistatic gene effect between chromosomes and changes in the degree of association of centromeric regions in interspecies hybrids. It is suggested that additional cytogenetic examination of blood lymphocytes and especially of testicles would help the understanding of the fertility barriers of hybrid males and would make a breeding programme for a stable crossbreed possible.     

Plasma growth hormone concentrations in female mithun (Bos frontalis) of different ages: relations with age and body weight

The role of growth hormone (GH) in postnatal somatic growth is well established. Its basal level and relation to growth performance in different age group mithun (Bos frontalis), a semiwild ruminant has not been characterized until now. To estimate the normal blood GH level and also to assess the influence of age and body weight (BW) on blood GH level in captive mithuns, a total of 65 female mithuns was divided into six age groups (group I, 0-6 months; group II, >6-12 months; group III, >1-2 years; group IV, >2-2.5 years; group V, >2.5-3.0 years and group VI, >3.0 years). Blood samples collected weekly for six consecutive weeks were assayed for GH. GH was also estimated in the samples collected from six growing mithuns at -60, -45, -30, -15, -10, -5 and 0 min prior to GH-releasing hormone (GHRH) administration for calculation of basal GH level and at 5, 10, 15, 30 min and thereafter at 15-min interval up to 8 h post-GHRH to assess blood GH response following GHRH administration in growing mithuns. For calculation of basal plasma GH in adult mithuns, GH was measured in blood samples collected at 30-min interval for 24 h from four animals. BW of all animals was recorded on two consecutive days per week and average of weekly BW was considered for growth rate calculation. It was found that both mean GH and GH per 100 kg BW between the age groups differ (p < 0.01). With increasing age and BW, GH and GH per 100 kg BW both decreased (p < 0.01). The age group with higher plasma GH and GH per 100 kg BW showed higher growth rates (r = 0.83 and 0.97 respectively). Interestingly, mean plasma GH for six consecutive weeks in all the groups showed much greater GH concentration (group I, 86.6 +/- 9.7 ng/ml to group VI 33.2 +/- 5 ng/ml) than reported in other species. Mean basal plasma GH calculated in growing and adult mithuns was 29.6 +/- 4.01 ng/ml and around 25 +/- 3.6 ng/ml respectively. The GH peak (444 +/- 21.3 ng/ml) was registered at 15 min post-GHRH administration in growing mithuns. In conclusion, age and BW influence plasma GH and GH per 100 kg BW but the latter is a better indicator of growth. The basal plasma GH and GH response to GHRH administration is six to eight and four to five times higher in mithun than in other species reported so far. An accurate assessment of the relationship between GH profiles and protein metabolism, proper receptor level study for GH action at the cellular level and the interaction of GH with other growth factors awaits better understanding of higher GH in this unique species.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Karyotyping, red blood cell and haemoglobin typing of the mithun (Bos frontalis), its wild ancestor and its hybrids

In Bhutan in remote parts of the Himalayas the mithun (Bos frontalis) has been used probably for centuries in an ingenious system for crossbreeding with domestic cattle which results in highly profitable hybrid females. The hybrid males are infertile and so far no stable crossbreed has been developed. Genetic analyses reported in this paper support the view that the gaur is the wild ancestor of the mithun. Both have only 58 chromosomes in contrast to 60 in cattle and also different, hitherto undescribed haemoglobins and blood groups which would justify the revision of the present classification of the subfamily Bovinae. If, with the aid of modern genetic methods, a stable crossbreed could be developed, dairy and beef production in Bhutan and many climatically similar areas could benefit greatly. Because of its remarkable size the mithun may, in the hands of enterprising breeders, also make a useful genetic contribution to beef production elsewhere.     

Effect of feeding Lagerstroemia speciosa and conventional fodder based rations on nutrient utilization,ruminal metabolites and body weight gain in mithun (Bos frontalis)

The aim of the present study was to evaluate the effect of feeding green fodder, rice straw and concentrate‐based total mixed rations (TMR) on dry matter (DM) intake (DMI), nutrient utilization, rumen fermentation patterns and body weight (BW) gain (BWG) in mithun (Bos frontalis) calves. In a randomized block design, male mithun calves (n = 18, 8–10 months of age, 121 ± 2 kg BW) were randomly divided into three experimental equal groups (six animals in each group) and fed isonitrogenous TMRs ad libitum for 120 days. The TMR₁ contained 30% Napier grass and 30% rice straw, TMR₂ contained 60% rice straw and TMR₃ contained 30% tree leaves (Lagerstroemia speciosa) and 30% rice straw (DM basis). All the TMRs contained 40% concentrate mixture (DM basis). The results indicated that the BWG, DMI and feed conversion efficiency were significantly (p < 0.01) increased with the inclusion of green fodder in TMRs. The apparent digestibility of DM, crude protein, ether extract, crude fibre and nitrogen free extract were also improved significantly (p < 0.01) with the inclusion of green fodder in TMRs. The higher concentration of total nitrogen and total volatile fatty acid in rumen liquor, but low ruminal pH were evident in animals fed green fodder supplemented TMRs. An increased (p < 0.01) molar proportion of acetic acid was evident in animals fed rice straw‐based TMR. In contrast, the molar proportion of propionic and butyric acids were significantly (p < 0.01) higher in animals fed green fodder supplemented TMRs. On the basis of higher DMI and higher daily BWG, it is concluded that Napier grass and L. speciosa tree leaves may be incorporated upto 30% (DM basis) in TMR of growing mithuns for feeding in complete confinement system.     

Effect of method and time of first colostrum feeding on serum immunoglobulin concentration, health status and body weight gain in mithun (Bos frontalis) calves

The effect of method and time of first colostrum feeding on the concentration of serum immunoglobulin (Ig) was evaluated in mithun (Bos frontalis) calves. The hypotheses were that the variable method and time of first colostrum feeding might affect the level of serum Ig and in turn the growth performance and health status of the claves during the early age. The newborn calves were randomly allotted to one of the four experimental groups - G-1: allowed to suckle the dam at own choice, G-2: separated immediately after birth and allowed to suckle the dam first at 6 h and then at own choice, G-3: bottle fed ad libitum colostrum of its own dam first at 6 h and then at 6-h intervals until 24 h, G-4: bottle fed ad libitum colostrum of its own dam within 1 h, at 6 h and then at 6-h intervals until 24 h. The concentrations of IgG, IgM, and IgA were lowest (p < 0.01) at birth and increased following colostrum feeding irrespective of the experimental group. Highest concentrations of all the Ig classes were observed at 12-24 h after birth. The concentrations then transiently decreased from day 7 to 14, and then steadily increased after day 28. The concentrations of IgG (p < 0.01) and IgA (p < 0.05) were higher in G-1 in relation to the other groups during the first week after birth. Similarly, higher concentration of IgA (p < 0.05) was found in G-1 in relation to the other groups during the rest of the experimental period. The apparent absorption efficiency of colostral IgG was higher (p < 0.05) in G-4 in relation to G-3. Growth rate and health status were not influenced by the method and time of first colostrum feeding. In conclusion, a 6-h delay in the first colostrum feeding reduced the level of serum Ig noticeably.     

Rumen parameters of yaks (Bos grunniens) and indigenous cattle (Bos taurus) grazing on the Qinghai‐Tibetan Plateau

Yaks and indigenous Qaidam cattle and cattle‐yak crosses (C × Y) graze on the Qinghai‐Tibetan Plateau (QTP) throughout the year, but yaks are raised at higher elevations than cattle. Yaks do not receive supplementary feed whereas cattle require supplementary feed during harsh winter. We hypothesized that yaks would cope with the severe conditions of the QTP better than cattle and utilize the pasture more efficiently. We also hypothesized that differences between species would be pronounced in winter, when conditions are particularly harsh. To test these hypotheses, seasonal rumen fluid parameters of yaks, C × Y and cattle (n = 3 for each) were examined. Rumen fluid was collected in summer and winter from each genotype 2, 5 and 12 hr after a day of grazing. Concentrations of total volatile fatty acid (VFA), acetate, propionate, isobutyrate and isovalerate were greater in yaks than in cattle in summer (p < 0.05), while propionate concentration was lower in yaks than in cattle in winter (p < 0.05). Concentrations of ammonia and urea were greater (p < 0.001) in yaks than in cattle (p < 0.001) in summer, whereas, concentrations of free amino acids (AA) were greater in cattle than in yaks in summer and winter (p < 0.001). Concentrations of total VFA, acetate, propionate and butyrate decreased linearly (p < 0.05), whereas concentrations of isobutyrate and isovalerate increased linearly for yak and C × Y with sampling time (p < 0.05) in summer. In summer, concentrations of isobutyrate and isovalerate were greater in yaks than in cattle (p < 0.05). In conclusion, rumen fermentation characteristics of yaks showed that they coped better than cattle or C × Y in the harsh climate as we hypothesized. However, in contrast to our hypothesis, this emerged only in summer, when pasture was plentiful and not in winter, when pasture was scarce.     

Post‐thawing effects of three cryopreservation diluents on Rusa deer (Rusa timorensis) spermatozoa

The aim of this study was to evaluate home‐made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES‐based, Tris‐based and Triladyl^® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post‐thawing and after 1‐hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post‐thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl^® and TES (p < 0.05) as compared with Tris (PM of Triladyl^® 14.7% vs. 3.2% TES and 2.5% Tris; VSL 56 for Triladyl^®, 59.2 for TES and 41.7 for Tris; LIN 45.6 for Triladyl^®, 52 for TES and 36.5 for Tris). Triladyl^® and TES extender led to better post‐thawing sperm parameters, but these preliminary results need to be verified through artificial insemination trials.     

大额牛溶菌酶(LYZ)基因序列测定和进化分析

[目的]为了对大额牛与其他物种间分子系统进化研究以及为大额牛群体遗传资源评价、保护与开发利用提供理论依据.[方法]本研究依据普通牛溶菌酶(lysozyme, LYZ)基因已知序列设计引物,用PCR的方法扩增并测定了大额牛LYZ基因的编码区核苷酸序列(444 bp),并与GenBank 上8个物种相应基因编码区核苷酸序列进行比对分析.[结果]大额牛与普通牛、人、猪、家鼠、沟鼠、猊猴、鸡、斑马鱼各物种在LYZ基因编码区核苷酸序列上同源性大小分别为99.3%、76.6%、 79.4%、81.8%、71.3%、68.2%、58.9%、50.5%,并分别采用邻接法(NJ)和最大简约法(MP)构建了大额牛与其他物种间分子进化树,并得到了基本一致的拓扑结构.系统发育分析发现大额牛与普通牛首先聚为一类,再与其他物种聚为一类.该系统聚类结果与动物学分类一致,表明LYZ基因适合于构建不同物种间系统进化树.[结论]本研究为进一步探索大额牛与近缘物种间分子进化,阐明大额牛的真正起源奠定一定实验基础.     

大额牛HSL基因外显子Ⅰ部分片段的序列测定及分析

对大额牛HSL基因外显子Ⅰ部分序列进行PCR扩增、测序及氨基酸预测,并同其它牛种的资料进行了比对分析,构建了分子系统进化树。结果表明:大额牛其核苷酸序列与牦牛、普通牛、瘤牛、水牛间的同源性分别为99.6%、99.4%、99.2%、97.0%。相应的氨基酸序列大额牛与水牛的同源性为97.6%;与普通牛、瘤牛、牦牛的同源性均为99.4%,仅在第33位有1个氨基酸变异,即大额牛为异亮氨酸,而其它3个牛种均为缬氨酸,这是由该基因片段的第97位碱基发生转换(A←→G)造成的。从分子系统进化树看,瘤牛和普通牛先聚为一类,再依次与牦牛、大额牛、水牛相聚,这与传统的牛种分类结果一致。     

Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen

Supplementing the extender with antioxidants with low molecular weight can enhance the quality of the post-thaw sperm during the freezing process. This study was aimed at determining the impacts of 3,4-dihydroxyphenyl glycol (DHPG) on the spermatozoa of the canine undergoing freeze-thawing process. In this study, 24 ejaculates were obtained from three mixed-breed dogs and were diluted in a Tris-based extender. The diluted semen was divided into aliquots for supplementation of 10, 30, 50 and 70 µg/ml of DHPG, control (without antioxidant) and control sham (DMSO). After being extended, the semen was equilibrated at a temperature of 4°C and then transferred to the straws and kept 4 cm above the liquid nitrogen for 20 min and was finally immersed in the liquid nitrogen. They were cryopreserved for seven days; then, sperm parameters including sperm motility evaluation, motility characteristics, viability, DNA and plasma membrane integrity, total antioxidant capacity (TAC), reduced glutathione content (GSH), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]) activity malondialdehyde (MDA) levels were evaluated. This study showed that spermatozoa cryopreservation with 50, 30 and 70 µg/ml of DHPG concentrations had better progressive motility, Curvilinear Velocity, Linearity, viability, intact plasma membrane and the levels of TAC, GPx and GSH were higher than the control group. The 50, 30 and 70 µg/ml of DHPG concentrations led to the significant decrease of DNA damage compared to the control group. Total motility, average path velocity, straight-line velocity and CAT activity were significantly improved in 30 and 50 µg/ml of DHPG concentrations, compared to the control group. Also, the 50 and 30 µg/ml of DHPG concentrations, decreased MDA levels compared to the other groups, significantly. In conclusion, our study showed that the addition 50 µg/ml of DHPG to the canine semen extender improved the semen characteristics and oxidative markers in the cryopreservation process.     

杂交牛(大额牛×云南黄牛)朊蛋白基因 的分子克隆及其序列分析

朊蛋白(prion protein,PRNP)基因编码朊蛋白,是引起疯牛病的主效基因。本研究利用PCR方法首次从杂交牛(大额牛×云南黄牛)基因组中扩增了PRNP基因,GenBank登录号为HQ875337。PCR产物直接双向测序表明,该序列包含杂交牛PRNP基因795 bp的开放阅读框(ORF),编码264个氨基酸前体蛋白。生物信息学分析结果发现,该蛋白包含1个信号肽、3个α螺旋、2个β折叠、6个八肽重复序列、1个疏水区域、1个二硫键和1个糖基磷脂酰肌醇锚定位点。与已报道的其他牛PRNP基因进行序列比对分析,核苷酸和氨基酸的同源性均在97%以上。