Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells

Spermatogonial stem cells (SSC) have important applications in domestic animal reproduction and advanced biotechnologies. Because differential plating is one of the most common methods used for SSC enrichment, the goal of this study was to compare three differential plating methods for the enrichment of bovine SSC. To achieve this goal, testicular parenchyma from pre‐pubertal calves was minced and single cells were obtained after two enzymatic digestions. We compared three coating methods for differential plating: laminin (20 ng/ml), BSA (0.05 mg/ml) and PBS. Cells were incubated at 37°C, 5% CO₂ in air for 15 min onto laminin‐coated dishes or 2 h onto BSA‐ or PBS‐coated dishes. Cell viability was assessed by trypan blue exclusion method. Recovered cells were analysed for the expression of SSC molecular markers by quantitative RT‐PCR (GFRA1, CXCR4, ITGA6, THY1) and flow cytometry (GFRA1, CXCR4 and ITGA6). Cells at time 0, adherent cells on laminin and non‐adherent cells from BSA and PBS groups had the same cell viability (p = 0.0655). GFRA1, CXCR4 and THY1 relative gene expression was higher (p = 0.0402, p = 0.0007, p = 0.0117, respectively) for non‐adherent cells selected in PBS group. Flow cytometry analysis revealed that the presence of GFRA‐positive (GFRA+) cells was higher in non‐adherent cells from BSA and PBS groups (p < 0.001). However, laminin‐adherent cells had higher number of ITGA6+ cells (p < 0.001) and lower presence of CXCR4+ cells (p = 0.0012). In conclusion, differential plating is an effective method for the enrichment of bovine undifferentiated spermatogonia and higher expression of SSC markers is obtained without laminin or BSA coating.     

Histamine production by transplantable argyrophilic gastric carcinoid of Praomys (Mastomys) natalensis

A transplantable argyrophilic gastric carcinoid of Praomys (Mastomys) natalensis contained appreciable amounts of histamine. Dialyzed, microsome-free supernatant of tumor tissue produced definite amounts of histamine in the presence of L-histidine and pyridoxal phosphate. These findings may relate to hypersecretion of gastric acid and formation of multiple ulcers in the stomach and duodenum of Mastomys bearing carcinoids.     

Improvement in In Vitro Fertilization Rate,Decrease in Reactive Oxygen Species and Spermatozoa Death Incidence in Rams by Dietary Fish Oil

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21‐day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer‐assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim‐up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10⁹/ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two‐cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O₂^?) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H₂O₂ rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen‐free radicals and the incidence of spermatozoa death.     

Effects of astaxanthin on Brachionus manjavacas (Rotifera) population growth

Astaxanthin is a red secondary carotenoid and powerful antioxidant that is used in aquaculture to enhance colour and improve fish health. Brachionid rotifers are often used as a live feed for larval fish, but do not contain endogenous carotenoids. However, they can be enriched with astaxanthin through their diet and transfer it to larval predators. When supplemented with 2 μg/ml astaxanthin oleoresin extracted from the green alga Haematococcus pluvialis, Brachionus manjavacas rotifer cultures reached significantly higher population densities and maintained them for longer. Furthermore, data are presented that exposure to oleoresin or pure astaxanthin enhances rotifer resistance to oxidative stress, a common cause for the collapse of rotifer mass cultures. Astaxanthin can be visualized in the gut of the rotifers, allowing the time course of uptake to be estimated by image analysis. Using this method, it was found that accumulation of astaxanthin in the rotifer gut saturates after 1.5 hr of exposure. The bioactive dose of astaxanthin oleoresin for rotifers was found to be 1–20 μg/ml. Astaxanthin concentration in rotifer tissues was measured using absorbance spectrophotometry. It was found that treating rotifers with 20 μg/ml for 24 hr; the concentration of astaxanthin absorbed into rotifer tissues was 2.6 mg/g. Overall, these experiments indicate that astaxanthin extracted from H. pluvialis can be used to improve the productivity and stability of rotifer mass cultures by increasing oxidative stress resistance and enhance the nutritional content of rotifers for larval fish.     

Use of GABA to enhance rotifer reproduction in enrichment culture

Gamma‐aminobutyric acid (GABA) has been shown to enhance the reproduction of the rotifer Brachionus plicatilis Muller in stressful culture conditions. During the enrichment of rotifers for feeding to marine fish larvae, they are usually stressed as a result of exposure to different marine oils and high population densities. This typically results in decreased rotifer survival, reproduction and swimming activity. In the present study, we used GABA to increase rotifer reproduction and the swimming activity of rotifers in enrichment cultures. GABA treatment 24 h before high density enrichment enhanced reproduction during enrichment culture, but not when carried out simultaneously with enrichment. Swimming activity was not significantly affected by GABA treatment 24 h before or simultaneously with nutrient enrichment.     

Effect of unionized ammonia, viscosity and protozoan contamination on the enzyme activity of the rotifer Brachionus plicatilis

Substrates that are cleaved to yield fluorescent products can be used to quickly quantify enzyme activity in vivo with image analysis or in vitro with fluorometry. This study was carried out to determine whether enzyme activity in rotifers is useful for assessing the physiological condition of rotifers. Neonates of Brachionus plicatilis Müller hatched from cysts were exposed to a concentration series of unionized ammonia ranging from 0 to 9.8 p.p.m., increasing seawater viscosity relative to 1.17 to control sea water by the addition of methyl cellulose and the addition of the protozoan Euplotes sp. to a density of 40 mL^?1. Rotifer glucosidase and esterase activities decreased with increasing unionized ammonia and viscosity respectively. Activities of glucosidase and phospholipase decreased with increasing protozoan contamination. There was a significant relationship between enzyme activities and rotifer population growth. In vivo activities of certain rotifer enzymes can therefore serve as biomarkers for the rapid assessment of environmental stressors in rotifer mass cultures.     

Giant Galápagos tortoises; molecular genetic analyses identify a trans-island hybrid in a repatriation program of an endangered taxon

Background

Giant Galápagos tortoises on the island of Española have been the focus of an intensive captive breeding-repatriation programme for over 35 years that saved the taxon from extinction. However, analysis of 118 samples from released individuals indicated that the bias sex ratio and large variance in reproductive success among the 15 breeders has severely reduced the effective population size (N _e ).

Results

We report here that an analysis of an additional 473 captive-bred tortoises released back to the island reveals an individual (E1465) that exhibits nuclear microsatellite alleles not found in any of the 15 breeders. Statistical analyses incorporating genotypes of 304 field-sampled individuals from all populations on the major islands indicate that E1465 is most probably a hybrid between an Española female tortoise and a male from the island of Pinzón, likely present on Española due to human transport.

Conclusion

Removal of E1465 as well as its father and possible (half-)siblings is warranted to prevent further contamination within this taxon of particular conservation significance. Despite this detected single contamination, it is highly noteworthy to emphasize the success of this repatriation program conducted over nearly 40 years and involving release of over 2000 captive-bred tortoises that now reproduce in situ. The incorporation of molecular genetic analysis of the program is providing guidance that will aid in monitoring the genetic integrity of this ambitious effort to restore a unique linage of a spectacular animal.

     

Effects of polyethylene colour and thickness on grass silage quality

The aim of the investigation was to determine the influence of variations in thickness and colour of agricultural plastic film on silage preservation conditions and grass silage quality. For this purpose, 30 cylindrical plastic containers (mini‐silos; 0·3 m³) were filled with chopped grass and covered with five films of different thickness and colour: 90 μm, white; 125 μm, green; 150 μm, black; 200 μm, green and 200 μm, white. Four replications of each film type were placed in the open air. Two replications were housed indoors and exposed to an ‘artificial sky’ in a test apparatus. The surface temperature of the films was found to be strongly dependent on film thickness and colour. Results of the chemical analysis of silages did not reveal any significant influence of the films. This was also the case when restricting the analysis to the uppermost silage layer. The results showed that under the conditions of this experiment, well preserved forage can be produced with films of differing colour, as well as of lesser thickness.     

ANGIOGRAPHIC APPEARANCE OF COMMON CAROTID COLLATERAL CIRCULATION IN THE GOAT RESULTING FROM PROSTHETIC GRAFT IMPLANTATION

Carotid arteriography was performed in six normal goats. The same procedure was performed on these six and 112 other goats at various time intervals after placement of an arterial graft in one or both carotid arteries. An arterial catheter was introduced into the femoral artery and advanced to the origin of the brachiocephalic trunk with the aid of image-intensified fluoroscopy. Hand-injection of the contrast medium resulted in complete opacification of both common carotid arteries in normal animals. Opacification of one or both of the vertebral arteries was usually seen in the goats with prosthetic grafts. The left vertebral artery was seen more often than the right, and when both vertebral arteries were seen together, the left was often larger. This angiographic method proved to be reliable for opacifying both common carotid arteries from their origin on the brachiocephalic trunk to the cranial cervical region. No clinical problems were associated with either the catheterization procedure or with the permanent ligation of the catheterized femoral artery.