Humanization of yeast to produce complex terminally sialylated glycoproteins

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.     

Extended day length in late winter/early spring,with a return to natural day length of shorter duration,increased plasma testosterone and sexual performance in rams with or without melatonin implants

Sixteen rams were used to quantify the effects of long days, imposed during late winter/early spring, with or without exogenous melatonin, on plasma testosterone concentrations and ram serving capacity. Rams were assigned to two groups: photoperiod‐treated rams (Artificial Photoperiod, AP; n = 8), exposed to 2 months of long days (16 hr of light/day) between 22 December and 22 February, and control rams (Natural Photoperiod, NP; n = 8). At the end of the long‐day period, AP rams were returned to the natural photoperiod, and each ram in the two groups either did (+M) or did not (‐M) receive three subcutaneous melatonin implants. Four groups were created as follows: AP+M (n = 4), AP‐M (n = 4), NP+M (n = 4) and NP‐M (n = 4). Thirty days after of the onset of photoperiodic treatment, AP rams (13.5 ± 2.8 ng/ml) had significantly (p < .05) lower testosterone levels than NP rams (36.7 ± 1.0), and similar differences were not apparent at the end of the photoperiod treatment. A month later, AP rams (24.3 ± 7.9) had higher (p < .10) testosterone levels than NP rams (13.1 ± 5.0), with no effect of melatonin treatment. Fifty days after melatonin implantations, rams were exposed for 20 min to three oestrous ewes. AP rams (2.50 ± 0.42) exhibited significantly (p < .05) more serves than did NP rams (1.11 ± 0.39), and melatonin treatment had no significant effect; however, the interaction between treatments was significant. Time to first serve was significantly (p < .05) shorter in AP (2.30 ± 1.20 min) than it was in NP rams (5.58 ± 0.68 min). In conclusion, exposure to 2 months of long days in late winter/early spring, with a return to natural day length of shorter duration, increased plasma testosterone concentrations and sexual performance in rams with or without exogenous melatonin. This particular management is an option if a non‐hormonal reproductive strategy is scheduled; yet, if the use of exogenous hormones is feasible, melatonin implants increase the mating efficiency of rams.     

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris–citrate–fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin–nigrosin) and plasma membrane integrity (Hypo‐osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona‐free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC‐processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid‐selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.     

不同区域奶牛子宫内膜炎病原菌的分离鉴定及分布特征

【目的】对中国6个区域奶牛子宫内膜炎病原菌的种类和区域分布特征进行研究,为筛选出最佳治疗药物以减少子宫内膜炎对畜牧业造成经济损失提供数据支持。【方法】分别从赤峰区域、张家口区域、潍坊区域、重庆区域、宝鸡区域和北京区域选择不同规模的奶牛养殖场中患子宫内膜炎的奶牛,用采样器每个区域采集60份(6个区域共计360份)患病牛的子宫内容物,通过革兰氏染色和生化鉴定的方法鉴定分离纯化的奶牛子宫内膜炎病原菌,并分析病原菌的分布特征。【结果】潍坊、赤峰、北京区域引起奶牛子宫内膜炎以单一感染为主。宝鸡、张家口、重庆区域引起奶牛子宫内膜炎以混合感染为主。赤峰、重庆、张家口、潍坊、宝鸡、北京区域引起奶牛子宫内膜炎的病原菌以革兰氏阳性菌为主。赤峰奶牛子宫内膜炎的主要致病菌是葡萄球菌。重庆奶牛子宫内膜炎的主要致病菌是链球菌。张家口奶牛子宫内膜炎的主要致病菌是大肠杆菌。潍坊奶牛子宫内膜炎的主要致病菌是链球菌。宝鸡奶牛子宫内膜炎的主要致病菌是葡萄球菌。北京奶牛子宫内膜炎的主要致病菌是蜡样芽孢杆菌。