Bovine foetal sex determination—Different DNA extraction and amplification approaches for efficient livestock production

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender‐specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell‐free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300–600 bp) in maternal circulation. The aim of this study was to assess this non‐invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non‐pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single‐tube extraction without silicone membranes and phenol–chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real‐time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single‐tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real‐time PCR test sensitivity in Group I was 90% and in Group II 91.6%.     

ACE inhibition in goats under fixed‐time artificial insemination protocol increases the pregnancy rate and twin births

To assess the effect of the angiotensin‐converting enzyme (ACE) inhibition on the efficiency of the fixed‐time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed‐time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0‐D2; 0.3 mg/kg/day in D3‐D6 and 0.4 mg/kg/day in D7‐D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ ATPase isoform in sturgeon sperm cryopreservation

Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane‐impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca²⁺ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S₁₅T₁₅) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L₁₅T₁₅) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L₁₅T₁₅ in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight‐line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.     

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post‐partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1‐ subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2‐ healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide‐binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post‐partum to measure mRNA abundance of TNF, interleukin‐1B (IL1B), interleukin‐6 (IL‐6), C‐X‐C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N‐acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N‐acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real‐time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro‐cytokine production and signalling, which may interfere with the onset and progression of inflammation.     

穗期玉米昼夜生理状态规律的遥感综合探测研究

针对QY、PRI、NDVI、水势分别从不同的方面反映玉米的生理状况,以甜糯二号玉米作为材料,通过田间试验测量玉米昼夜4个参数值,研究了QY、PRI、NDVI、水势4个参数的昼夜变化,并且分析它们之间的相关性。结果表明,QY、水势、PRI三者具有很强的相关性,并且能建立回归模型表示它们之间的联系。     

Differential Cl−/Salt Tolerance and NaCl‐Induced Alternations of Tissue and Cellular Ion Fluxes in Glycine max,Glycine soja and their Hybrid Seedlings

The salt‐sensitive Glycine max N23674 cultivar, the salt‐born Glycine soja BB52 population, and their hybrid 4076 strain (F₅) selected for salt tolerance generation by generation were used as the experimental materials in this study. First, the effects of NaCl stress on seed germination, tissue damage, and time‐course ionic absorption and transportation were compared. When qualitatively compared with seed germination appearance in culture dishes, and tissue damages on roots or leaves of seedlings, or quantitatively compared with the relative salt injury rate, the inhibition on N23674 was all the most remarkable. After the exposure of 140 mm NaCl for 1 h, 4 h, 8 h, 12 h, 2 days and 4 days, the content of Cl^? gradually increased in the roots and leaves of seedlings of BB52, 4076 and 23674. Interestingly, the extents of the Cl^? rise in roots of the three experimental soybean materials were BB52 > 4076 > N23674, whereas those in leaves were just on the contrary. Secondly, by using the scanning ion‐selective electrode technique (SIET), fluxes of Na⁺ and Cl^? in roots and protoplasts isolated from roots and leaves were also investigated among the three experimental soybean materials. After 140 mm NaCl stress for 2, 4 and 6 days, and when compared with N23674, slighter net Cl^? influxes were observed in root tissue and protoplasts of roots and leaves of BB52 and 4076 seedlings, especially at the cellular protoplast level. The results indicate that with regard to the ionic effect of NaCl stress, Cl^? was the main determinant salt ion for salt tolerance in G. soja, G. max and their hybrid, and the difference in their Cl^?/salt tolerance is mainly attributed to the capacity of Cl^? restriction to the plant above‐ground parts such as leaves.     

Pedotransfer functions to assess adsorbed phosphate using iron oxide content and magnetic susceptibility in an Oxisol

Adsorbed phosphate in soils can be chemically extracted; however, this process is both time‐consuming and not cost‐effective if large numbers of samples have to be analysed. Indirect assessment of adsorbed phosphate by pedotransfer functions (PTFs) can help optimize fertilizer strategies. This study aimed to evaluate the spatial variability of adsorbed phosphate (P_ads), iron oxides and magnetic susceptibility (MS) in oxisols and to calibrate PTFs to predict P_ads. A total of 308 soil samples were collected from Hapludox and Eutrudox soils formed from sandstone in Brazil. The contents of clay (196–607 g/kg), iron oxides (40–165 g/kg), MS (1.2–29 × 10^?6 m³/kg) and P_ads (327–842 mg/kg) were in the range of typical values for these highly weathered soils. This study showed that the attributes studied were spatially dependent. Geomorphic surfaces enabled understanding of spatial variability and helped to develop a more efficient sampling scheme to calibrate PTFs. Moreover, the adsorbed phosphate in these oxisols could be predicted by a PTF using iron oxides and MS as predictors. The MS attribute enabled the most accurate prediction (concordance coefficient = 0.95, root‐mean‐square error = 46 mg/kg and relative improvement in root‐mean‐square error = ?4.12) of spatial variability through PTF compared to other predictors.     

Abscisic Acid May Alter the Salicylic Acid‐Related Abiotic Stress Response in Maize

The effect of 0, 0.05 or 0.1 mm abscisic acid treatment on chilling tolerance and salicylic acid‐related responses was investigated in young maize seedlings (Zea mays L., hybrid Norma). Although the pre‐treatment of maize seedlings with abscisic acid slightly decreased the chlorophyll content, it also reduced the level of chilling injury caused by 6 days of cold treatment at 5 °C. Under normal growth conditions, increased levels of bound salicylic acid and of bound ortho‐hydroxycinnamic acid were observed in the leaves during abscisic acid treatment. In the roots, abscisic acid did not affect the free and bound salicylic acid levels, but increased the amount of free and bound ortho‐hydroxycinnamic acid. The activity of glutathione‐S‐transferase increased on the 3rd day of abscisic acid treatment, whereas it did not change when followed by cold stress, compared with the control leaves. In the roots, the activities of glutathione reductase, glutathione‐S‐transferase and ascorbate peroxidase increased during the abscisic acid treatment, and those of glutathione‐S‐transferase and ascorbate peroxidase were also stimulated when abscisic acid pre‐treatment was followed by cold stress, compared with the control roots. Our results suggest that an overlap may exist between the abscisic acid‐induced cold acclimation and the salicylic acid‐related stress response.     

5‐Aminolevulinic Acid Activates Antioxidative Defence System and Seedling Growth in Brassica napus L. under Water‐Deficit Stress

The present study assesses the effects of 5‐aminolevulinic acid (ALA, 0, 0.1, 1 and 10 mg l^?1) on the growth of oilseed rape (Brassica napus L. cv. ZS758) seedlings under water‐deficit stress induced by polyethylene glycol (PEG 6000, 0 and ?0.3 MPa). Water‐deficit stress imposed negative effects on seedling growth by reducing shoot biomass, cotyledon water potential, chlorophyll content and non‐enzymatic antioxidants (glutathione and ascorbic acid) levels. On the other hand, water‐deficit stress enhanced the malondialdehyde (MDA) content, reactive oxygen species (ROS) production, enzymatic antioxidants activities, reduced/oxidized glutathione ratio (GSH/GSSG) and reduced/oxidized ascorbic acid (ASA/DHA) ratio in seedlings. Application of ALA at lower dosages (0.1 and 1 mg l^?1) improved shoot weight and chlorophyll contents, and decreased MDA in rape seedlings, whereas moderately higher dosage of ALA (10 mg l^?1) hampered the growth. The study also indicated that 1 mg l^?1 ALA improved chlorophyll content, but reduced MDA content and ROS production significantly under water‐deficit stress. Lower dosages of ALA (0.1 and 1 mg l^?1) also enhanced GSH/GSSG and ASA/DHA as compared to the seedlings under water‐deficit stress. The antioxidant enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase and superoxide dismutase) enhanced their activities remarkably with 1 mg l^?1 ALA treatment under water‐deficit stress. It was also revealed that 1 mg l^?1 ALA treatment alone induced the expression of APX, CAT and GR substantially and under water‐deficit stress conditions ALA treatment could induce the expression of POD, CAT and GR to a certain degree. These results indicated that 0.1–1 mg l^?1 ALA could enhance the water‐deficit stress tolerance of oilseed seedlings through improving the biomass accumulation, maintaining a relative high ratio of GSH/GSSG and ASA/DHA, enhancing the activities of the specific antioxidant enzymes and inducing the expression of the specific antioxidant enzyme genes.