Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.     

用牦牛睾丸细胞生产羊传染性脓疱皮炎疫苗效果观察

为满足生产批量化要求，用牦牛睾丸细胞代替犊牛睾丸细胞，接种G0-BT弱毒株，收获，加适当保护剂，经冷冻真空干燥制备羊传染性脓疱皮炎疫苗。结果半成品及成品检验均符合《规程》标准，取得了良好的效果。     

The Oxidative Stress,Antioxidant Profile and Acid–base Status in Preterm and Term Canine Neonates

During the initiation of neonatal pulmonary respiration, there is an exponential increase in reactive oxygen species that must be scavenged by antioxidant defences. However, neonate and preterm newborns are known to possess immature antioxidant mechanisms to neutralize these toxic effects. The purposes of this study were to compare the development of antioxidant system between preterm and term canine neonates and to evaluate the magnitude of acid–base balance during the initial 4 h of life. A prospective study was conducted involving 18 neonatal puppies assigned to Term Group (63 days of gestation; n = 5), Preterm‐57 Group (57 days of gestation; n = 8) and Preterm‐55 Group (55 days of gestation; n = 5). Neonates were physically examined through Apgar score and venous haemogasometry within 5 min, 2 and 4 h after birth. No difference on amniotic fluid and serum superoxide dismutase (SOD), glutathione peroxidase (GPx) and the marker of oxidative stress (thiobarbituric acid reactive substances; TBARS) was verified. Irrespective of prematurity, all neonates presented low vitality, hypothermia, acidosis, hypoxaemia and hypercapnia at birth. However, term puppies clinically evolved more rapidly than preterm newborns. During the course of the study, premature neonates presented more severe complications, such as prolonged hypoxaemia and even death. In conclusion, premature puppies have no signs of immature enzymatic mechanisms for controlling oxidative stress, although SOD and GPx may participate in achieving acid–base balance. Aside from initial unremarkable symptoms, premature puppies should be carefully followed up, as they are at high risk of succumbing to odds of prematurity.     

Involvement of PAPP‐A and IGFR1 in Cystic Ovarian Disease in Cattle

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors, such as members of the insulin‐like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy‐associated plasma protein A (PAPP‐A). The aim of this study was to determine IGFR1 and PAPP‐A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP‐A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP‐A present in the follicular fluid from these follicles showed no differences. Although no PAPP‐A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.     

Effects of aggregate size,soil compaction,and bovine urine on N2O emissions from a pasture soil

The dominant N₂O emission source in New Zealand, calculated using the Intergovernmental Panel on Climate Change methodology, is agricultural soils. The largest source of N₂O emissions in New Zealand occurs as a result of excreta deposition onto pasture during grazing. There is a dearth of studies examining the effect of soil compaction and soil aggregate size on N₂O emissions from urine patches in grazed pastures. In this study, we repacked soil cores with four different soil aggregate sizes (<1.0–5.6 mm diameter), applied bovine urine, and then subjected the soil cores to four levels of soil compaction. Fluxes of N₂O were monitored for 37 days after which soil cores were allowed to dry out prior to a rewetting event. There was an interaction between aggregate size and soil compaction with the cumulative loss of N₂O over the first 37 days ranging from 0.3% to 9.6% of the urine-N applied. The highest N₂O emissions occurred from the smallest and most compacted aggregates. Even under the highest levels of compaction the N₂O loss from the large aggregates (4.0–5.6 mm diameter) was <1% of the urine-N applied. Reasons for the observed differences in the N₂O flux from the different-sized aggregates included varying gas diffusivities and higher rates of denitrification in the smallest aggregates, as evidenced by the disappearance of nitrate.     

Forest–atmosphere carbon dioxide exchange in eastern Siberia

We investigated the daily exchange of CO₂ between undisturbed Larix gmelinii (Rupr.) Rupr. forest and the atmosphere at a remote Siberian site during July and August of 1993. Our goal was to measure and partition total CO₂ exchanges into aboveground and belowground components by measuring forest and understory eddy and storage fluxes and then to determine the relationships between the environmental factors and these observations of ecosystem metabolism. Maximum net CO₂ uptake of the forest ecosystem was extremely low compared to the forests elsewhere, reaching a peak of only ∼5 μmol m⁻² s⁻¹ late in the morning. Net ecosystem CO₂ uptake increased with increasing photosynthetically active photon flux density (PPFD) and decreased as the atmospheric water vapor saturation deficit (D) increased. Daytime ecosystem CO₂ uptake increased immediately after rain and declined sharply after about six days of drought. Ecosystem respiration at night averaged ∼2.4 μmol m⁻² s⁻¹ with about 40% of this coming from the forest floor (roots and heterotrophs). The relationship between the understory eddy flux and soil temperature at 5 cm followed an Arrhenius model, increasing exponentially with temperature (Q₁₀∼2.3) so that on hot summer afternoons the ecosystem became a source of CO₂. Tree canopy CO₂ exchange was calculated as the difference between above and below canopy eddy flux. Canopy uptake saturated at ∼6 μmol CO₂ m⁻² s⁻¹ for a PPFD above 500 μmol m⁻² s⁻¹ and decreased with increasing D. The optimal stomatal control model of Mäkelä et al. (1996) was used as a `big leaf' canopy model with parameter values determined by the non-linear least squares. The model accurately simulated the response of the forest to light, saturation deficit and drought. The precision of the model was such that the daily pattern of residuals between modeled and measured forest exchange reproduced the component storage flux. The model and independent leaf-level measurements suggest that the marginal water cost of plant C gain in Larix gmelinii is more similar to values from deciduous or desert species than other boreal forests. During the middle of the summer, the L. gmelinii forest ecosystem is generally a net sink for CO₂, storing ∼0.75 g C m⁻² d⁻¹.     

Production, behaviour and fertility of Merino wethers, hemi-castrates with reduced testicular parenchyma and induced cryptorchids

Greasy wool production, body weight, behaviour and fertility of male, fine wool Merino sheep were studied from 10 to 21 months of age to assess their suitability for wool production. The males were either castrated (wethers), or were hemi-castrated with a vasectomy and reduction (either partially or in full) of the parenchyma of the retained testicle, or the testicles pushed up into the ingulnal canal and the scrotum shortened (induced cryptorchids). There were significant differences among all groups in body weight, with increases being associated with the larger amounts of testicular tissue present. Fertility was low in the induced cryptorchids at 19 months of age. The hemi-castrates with reduced testicular parenchyma were infertile. The hemi-castrates with reduced testicular parenchyma had significantly heavier greasy fleece weights than the wethers and induced cryptorchids, which had similar fleece weights. The wether group suffered a higher prevalence of posthitis in spring and autumn than the hemi-castrate or induced cryptorchid groups. Those hemi-castrates with the largest amount of testicular parenchyma retained and the induced cryptorchids exhibited masculine behaviour patterns and had an increased incidence of fly strike to the head. The principle of hemi-castration and reduction of the testicular parenchyma is shown to be applicable in wool producing flocks with the potential to increase greasy wool production while minimising the management and marketing limitations previously associated with induced cryptorchids.     

Sperm Morphology Assessment in Captive Neotropical Primates

The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO‐2010) standards and Sperm Deformity Index (SDI) criteria, objective computer‐assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO‐2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.     

Genomic Imprinting in Canis familiaris

For the vast majority of mammalian genes, maternally- and paternally-derived alleles behave identically and are either expressed or repressed, regardless of whether they were inherited from egg or sperm. For imprinted genes, however, this is not the case. The alleles of imprinted genes are epigenetically modified in a parent-of-origin-specific manner and, as a consequence, maternally- and paternally-derived alleles behave differently. Typically one allele is expressed while the other is silent. Although relatively few in number, imprinted genes are the focus of intensive study, as they have important roles in embryonic development. Abnormal expression of imprinted genes results in growth disorders and is implicated in several clinical conditions. Most studies of imprinted genes have been performed in rodents or primates, with limited studies in other mammals such as bovine and opossum. We have recently demonstrated the existence of imprinted genes in the canine, by showing that the canine insulin-like growth factor 2 receptor gene ( IGF2R ) is monoallelically expressed, with predominant expression of the maternally-derived allele and repression of the paternally-inherited allele. Our ultimate goal is to characterize all imprinted genes in the canine, and to understand how they contribute to canine reproduction, development and disease. Such knowledge will be vital for optimizing the success of most reproductive strategies in the canine.     

Concentrations of Prolactin, LH, Testosterone, TSH and Thyroxine in Normospermic Dogs of Different Breeds

Concentrations of prolactin (PRL), LH, testosterone (T), TSH and thyroxine (T₄) were determined before and at 20, 120 and 180 min after a single iv injection of thyrotropin-releasing hormone (TRH) in eight Beagles, eight Fox Terriers, six Labrador Retrievers and five Great Danes that were normospermic. Mean basal PRL concentrations were lower in the Fox Terriers compared with the Great Danes (p < 0.05). Mean LH concentrations were higher in the Fox Terriers than in the Beagles, and T was lower in the Fox Terriers at some times but not others (p < 0.05). Thyroid Stimulating Hormone (TSH) concentrations did not differ among breeds, while mean basal T₄ values were lower in Fox Terriers compared with Labrador Retrievers and Great Danes (p < 0.05). Stimulation of T₄ secretion 120 and 180 min after iv TRH injection was most pronounced in the Beagles and less in the Fox Terriers (p < 0.05). The results of the present study indicate that potential breed differences in circulating concentrations of PRL, LH, T, TSH and T₄ in male dogs with apparently normal fertility can be encountered, but further studies are needed to determine whether the observed differences are typical features of these breeds, reflect subsets of dogs within breeds, or are in part because of possible uncontrolled parameters such as sample timing, ambient photoperiod, housing conditions or diet.