Comparison of Membrane‐Permeant Fluorescent Probes for Sperm Viability Assessment in the Ram

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR‐14; Hoechst‐33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR‐14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane‐affected sperm (semen treated with three cycles of freezing to ?20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma‐intact sperm determined by acridine orange and SYBR‐14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.     

Assessing regional species pools for restoration programs in Spain

The relationship between current and potential distribution in 34 main Spanish forest tree species (data from the Third Spanish Forest Inventory) was determined using a Maximum Entropy functional approximation with climatic data as predictive variables. A method for detecting regional species pools at two different scales: biogeoclimatic classes (CLATERES classification), and forest landscape types (WWF classification) has been proposed. Then, the Absence percentage for a species (i.e. the proportion of landscapes types or biogeoclimatic classes in which the species is included in the regional species pool but is actually not present) was determined. Results show higher figures of Potential Species Richness in the Pyrenees and the Cantabrian Range, while inland or coastal Mediterranean semiarid landscapes have lower figures. Using a classification based on biogeoclimatic variables (CLATERES) improves precision when estimating Absent Species Richness. Absence percentage is zero or close to zero for five species (Pinus uncinata, Quercus robur, Quercus ilex, Quercus humilis and Juniperus communis), while for other six species (Acer pseudoplatanus, Fraxinus angustifolia, Alnus glutinosa, Populus alba, Sorbus aucuparia and Pinus pinea) the figures are higher than 0.6, which means the species is absent in more than 60 % of the landscapes or biogeoclimatic classes that it could inhabit. The relationships between tree life traits and the absence of species from the ecosystems studied is slight but non-dominant species, species not subjected to forest management, or zonal species are less widely distributed that their climatic potentiality indicates.     

The Effects of Sample Size on the Outcome of Ovarian Tissue Cryopreservation

Cryopreservation of ovarian tissue is known to affect follicular survival. Several variables may be responsible for this. Little attention has focused on the effect of the size of the fragment to be cryopreserved. This study was conducted to assess the effect of the size of the tissue on follicular histology after freezing with 1,2-propanediol. Histological evaluations were performed of control and cryopreserved tissue. Fragments were cut 10 × 3 × 2 mm³ (2 mm group) or 10 × 3 × 4 mm³ (4 mm group). Percentages of normal follicles in control fragments cut into 2 and 4 mm slices were 56% and 34%, respectively. The relative risks to obtain normal follicles in the 2 mm and the 4 mm fragments after cryopreservation were 0.63 and 0.47, respectively. Freezing reduced follicle survival to a significantly greater extent in the larger tissue fragments. There is an increased risk of damage to primary and primordial follicles, when the tissue slices are cut with all dimensions larger than 2 mm.     

Association with TGF‐β1 Gene Polymorphisms and Reproductive Performance of Large White Pig

As a multifunctional cytokine, transforming growth factor‐beta1 (TGF‐β1) was detected in the utero‐placental interface during early pregnancy in the pig and believed to enhance trophoblast attachment to the endometrium. In this experiment, we selected TGF‐β1 as the candidate gene affecting litter size in pigs. Four polymorphic loci of TGF‐β1 gene were found by PCR‐SSCP (single‐strand conformation polymorphism) in Large white sows (n = 567): C→T mutation at 33nt in the intron 4; G→A mutation at 179nt in the intron 6; C→T mutation at 1043nt in the intron 6; GG→AA linkage mutations at 2490nt and 2494nt respectively. We haplotyped these SNPs as: CGCAA (denote as P) and TATGG (denote as K). The effects of three haplotypic combinations (HCs) of PP, PK and KK on litter sizes were investigated by a linear model. It was found that for the first parity litters, the least squares mean for total number born (TNB) of KK was 1.02 piglets/litter, higher than that of PK (p < 0.05), 0.49 piglets/litter higher than that of PP (p > 0.1). There were no significant differences between HCs on the second parity. The result indicated that KK HCs was significantly associated with pig litter size.     

The Influence of Powdered Coconut Water (ACP‐318®) in In Vitro Maturation of Canine Oocytes

The objective of this study was to determine the influence of powdered coconut water (ACP‐318^®) diluted in high glucose (11.0 mm ) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Cumulus oocyte complexes (COCs) (n = 632) were randomly allocated into three experimental groups named as group 1 (control group), group 2 (5% powdered coconut water) and group 3 (10% powdered coconut water). The percentage of meiotic resumption (MR) (GVBD to MII) was 39.1% (81/207), 50.2% (108/215) and 46.6% (98/210) for groups 1, 2 and 3 respectively (p < 0.05). There were no differences in MR rates among groups 2 and 3. The medium with ACP‐318^® slightly enhanced the nuclear maturation of canine oocytes when a comparison was established with rates of maturation exhibited by oocytes in the experimental group 1 without ACP‐318^® (p < 0.05). The results suggest that oocytes’ nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water. Further investigation must be performed for a better understanding of powdered coconut water influence in cellular events during IVM of dog oocytes.     

Comparison of the omp I gene of Chlamydia psittaci between isolates in Victorian koalas and other animal species

Objective The objective of this study is to compare the strain of chlamydia causing genital infection in koalas from Victoria with isolates from other animal species.
Design Polymerase chain reaction and restriction enzyme analysis has been used to compare various Chlamydia psittaci isolates from a range of animals and disease syndromes. The isolates used in this study include isolates from three birds, three from aborted sheep, one from polyarthritis, one from bovine abortion, one from feline pneumonitis, three porcine isolates from faeces, polyarthritis and abortion, and three urogenital isolates from Victorian koalas.
Procedure Two polymerase chain reactions were performed, each amplifying a different region of the omp I gene. The first polymerase chain reaction amplified a 144 bp segment of the gene which was then digested with the restriction enzyme Eco R I. The second polymerase chain reaction amplified a larger 1070 bp region of the omp I gene which was digested with two restriction enzymes Alu I and Nde II.
Results and conclusions The results obtained have confirmed that variation in DNA sequence of various animal chlamydia isolates does occur. They have also shown that it is possible to classify isolates, based on their restriction enzyme profiles, into distinct groups.     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

磷酸泰乐菌素颗粒剂生物效价测定方法的建立及验证

建立了磷酸泰乐菌素颗粒剂的生物效价测定方法，并对新建立的测定方法进行了验证，验证内容包括：方法对照试验、精密度试验、线性及线性范围试验、回收率试验。验证结果证明新方法能准确测定磷酸泰乐菌素颗粒剂的效价单位。