Production of Oocytes of Nile Tilapia (Oreochromis niloticus) for In Vitro Fertilization via Hormonal Treatments

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin‐releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post‐hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.     

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Antioxidants are known to prevent the reactive oxygen species (ROS)‐mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid–protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll^®. Sperm pellet was resuspended in soya lecithin–Tris–fructose diluent (400 × 10⁶ cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3–5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo‐osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X‐100 were resolved by SDS‐PAGE and quantified using Quantity One software (Bio‐Rad, USA). The rapid motility, linearity and straight‐line velocity (VSL) were found significantly (p < .05) higher in the antioxidant‐treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant‐treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant‐treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant‐treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.     

Fertility of Angus cross beef heifers after GnRH treatment on day 23 and timing of insemination in 14‐day CIDR protocol

This study compared artificial insemination pregnancy rate (AI‐PR) between 14‐day CIDR‐GnRH‐PGF2α‐GnRH and CIDR‐PGF2α‐GnRH synchronization protocol with two fixed AI times (56 or 72 hr after PGF2α). On day 0, heifers (n = 1311) from nine locations assigned body condition score (BCS: 1, emaciated; 9, obese), reproductive tract score (RTS: 1, immature, acyclic; 5, mature, cyclic) and temperament score (0, calm; and 1, excited) and fitted with a controlled internal drug release (CIDR, 1.38 g of progesterone) insert for 14 days. Within herd, heifers were randomly assigned either to no‐GnRH group (n = 635) or to GnRH group (n = 676), and heifers in GnRH group received 100 μg of GnRH (gonadorelin hydrochloride, IM) on day 23. All heifers received 25 mg of PGF2α (dinoprost, IM) on day 30 and oestrous detection aids at the same time. Heifers were observed for oestrus thrice daily until AI. Within GnRH groups, heifers were randomly assigned to either AI‐56 or AI‐72 groups. Heifers in AI‐56 group (n = 667) were inseminated at 56 hr (day 32 PM), and heifers in AI‐72 group (n = 644) were inseminated at 72 hr (day 33 AM) after PGF2α administration. All heifers were given 100 μg of GnRH concurrently at the time AI. Controlling for BCS (p < .05), RTS (p < .05), oestrous expression (p < .001), temperament (p < .001) and GnRH treatment by time of insemination (p < .001), the AI‐PR differed between GnRH treatment [GnRH (Yes – 60.9% (412/676) vs. No – 55.1% (350/635); p < .05)] and insemination time [AI‐56 – 54.6% (364/667) vs. AI‐72 – 61.8% (398/644); (p < .01)] groups. The GnRH treatment by AI time interaction influenced AI‐PR (GnRH56 – 61.0% (208/341); GnRH72 – 60.9% (204/335); No‐GnRH56 – 47.9% (156/326); No‐GnRH72 – 62.8% (194/309); p < .001). In conclusion, 14‐day CIDR synchronization protocol for FTAI required inclusion of GnRH on day 23 if inseminations were to be performed at 56 hr after PGF2α in order to achieve greater AI‐PR.     

Estradiol stimulates glycogen synthesis whereas progesterone promotes glycogen catabolism in the uterus of the American mink (Neovison vison)

Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E₂) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre‐embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P₄) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E₂, P₄ or vehicle (controls) for 3 days and uteri collected 24 h (E₂, P₄ and vehicle) and 96 h (E₂) later. To evaluate E₂ priming, mink were treated with E₂ for 3 days, then P₄ for an additional 3 days (E₂→P₄) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E₂ + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E₂+24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P < 0.05). Treatment as E₂→P₄ reduced glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose‐6‐phosphatase) gene expression and amount of phospho‐glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E₂→P₄ increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E₂ promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P₄ that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre‐embryonic survival and implantation.     

The significance of the zoosporangial stage in the life cycle of the plasmodiophorales

Samenvatting Bij de Plasmodiophorales onderscheidt men het zoösporangium- en het rustsporenstadium. Het zoösporangiumstadium wordt gekenmerkt door de aanwezigheid van zoösporangia, vaak in wortelharen en andere schorsepidermiscellen; het rustsporenstadium door het tot ontwikkeling komen van rustsporen, dikwijls in gehypertrofieerde delen van de waardplant. Sinds de ontdekking van het zoösporangiumstadium is nimmer zekerheid verkregen over de vraag of zoösporen uit de zoösporangia bij herinfectie wederom zoösporangia kunnen geven. Het alternatief zou zijn dat de zoösporen uit de zoösporangia de levenscyclus vervolgen en tot de ontwikkeling van rustsporen leiden.Een infectieproef metSpongospora subterranea bij tomateplanten heeft nu uitgewezen dat de eerste veronderstelling juist is; zoösporen uit de zoösporangia geven bij herinfectie inderdaad weer zoösporangia. Het rustsporenstadium komt waarschijnlijk tot ontwikkeling na herinfectie door een zygote, welke ontstaat indien twee als gameten fungerende zoösporen versmelten. Deze opbouw van de levenscyclus (fig. 1) geeft de schimmel de mogelijkheid tot een sterke vegetatieve vermeerdering in het zoösporangiumstadium.     

珠芽尖距紫堇 Corydalis sheareri对两种鳞翅目昆虫拒食和拒产卵活性的初步研究

分别用液—液分配法和柱层析法对珠芽尖距紫堇Corydalis sheareri 全株的氯仿粗提物进行了分离, 以求获得对甘蓝害虫有拒食活性的物质。液—液分配法中, 苯提取物的拒食活性最高, 在50 倍稀释浓度下, 小菜蛾Plutellaxy lostella 和菜粉蝶Pierisrapae 幼虫对其拒食率分别为97.7% 和97.3%。苯提取物还显示了很高的拒产卵活性。通过硅胶柱层析获得了相对较纯的提取物, 其中活性最高的成分在200 倍的稀释浓度下对菜粉蝶的拒食率为93.0%。根据现有资料对上述分离物的化学本质进行了探讨。     

Detection of hypoglycin A in the seeds of sycamore (Acer pseudoplatanus) and box elder (A. negundo) in New Zealand; the toxin associated with cases of equine atypical myopathy

CASE HISTORY AND CLINICAL FINDINGS: During April and May 2014 four horses aged between 5 months and 9 years, located in the Canterbury, Marlborough and Southland regions, presented with a variety of clinical signs including recumbency, stiffness, lethargy, dehydration, depression, and myoglobinuria suggestive of acute muscle damage. Two horses were subjected to euthanasia and two recovered. In all cases seeds of sycamore maple (Acer pseudoplatanus) or box elder (A. negundo) were present in the area where the horse had been grazing.

LABORATORY INVESTIGATION: The samaras (seeds) of some Acer spp. may contain hypoglycin A, that has been associated with cases of atypical myopathy in Europe and North America. To determine if hypoglycin A is present in the samaras of Acer spp. in New Zealand, samples were collected from trees throughout the country that were associated with historical and/or current cases of atypical myopathy, and analysed for hypoglycin A. Serum samples from the four cases and four unaffected horses were analysed for the presence of hypoglycin A, profiles of acylcarnitines (the definitive diagnosis for atypical myopathy) and activities of creatine kinase and aspartate aminotransferase.Markedly elevated serum activities of creatine kinase and aspartate aminotransferase, and increased concentrations of selected acylcarnitines were found in the case horses. Hypoglycin A was detected in the serum of those horses but not in the healthy controls. Hypoglycin A was detected in 10/15 samples of samaras from sycamore maple and box elder from throughout New Zealand.

DIAGNOSIS: Cases of atypical myopathy were diagnosed on properties where samaras containing hypoglycin A were also found.

CLINICAL RELEVANCE: Sycamore and box elder trees in New Zealand are a source of hypoglycin A associated with the development of atypical myopathy. If pastured horses present with clinical and biochemical signs of severe muscle damage then the environment should be checked for the presence of these trees. Horses should be prevented from grazing samaras from Acer spp. in the autumn.