Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

Conception rates to fixed-time artificial insemination of two oestrus synchronisation programmes in dairy heifers

AIM: To evaluate the conception rate to fixed-time artificial insemination (FTAI) of two oestrus synchronisation programmes in dairy heifers on eight farms over 2 years.

METHODS: The study was conducted in 2008 and 2010 on eight farms near Palmerston North, New Zealand. Nulliparous Friesian and Friesian×Jersey heifers (13–15 months of age) were randomly allocated to one of two oestrus synchronisation programmes. Group 1 (GPG+P4; n=330), received gonadotrophin-releasing hormone (GnRH) I/M on Day 0, a progesterone (P4)-releasing intravaginal device from Days 0–7, prostaglandin F_2α (PGF) I/M on Day 7 and a second dose of GnRH at the time of FTAI on Day 9. The second group (P4+PGF; n=343) received a P4-releasing intravaginal device from Days 0–7, PGF on Day 6 and FTAI on Day 9. Pregnancy was diagnosed from Days 42–52 by transrectal ultrasonography.

RESULTS: The overall conception rate was 52.4% and 54.8% for the GPG+P4 and P4+PGF groups, respectively. The odds of conception for the two treatments were not different (OR=0.90; 95% CI=0.67–1.23), nor was there any difference between groups in different years (p=0.58). Farm affected conception rate (p=0.002), but there was no interaction with treatment (p=0.92) .

CONCLUSIONS: This study has shown that an alternative synchronisation programme can produce similar results in terms of conception rate to the GPG+P4 treatment, currently commonly used in heifers. More research is required to establish whether other modifications to the GPG+P4 programme can produce similar results at lower costs, and to identify and quantify farm factors which affect the economic benefit of heifer synchronisation.

CLINICAL RELEVANCE: This study indicated that synchronising heifers with P4 and PGF resulted in conception rates equivalent to those resulting from a GPG+P4 treatment, but with reduced drug costs. However, because heifers in the GPG+P4 group received the second GnRH injection at the time of AI, they needed only three yardings as opposed to the four required for the heifers treated with P4 and PGF. Thus, the choice of programme for an individual farm will depend on that farm's circumstances, in particular the cost of yarding the heifers.     

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.     

Extraction of seed oil from Diospyros lotus optimized using response surface methodology

Oil from seeds of Diospyros lotus was extracted using a conventional method with two different solvents:hexane and petroleum ether. A central composite design with response surface methodology were used to optimize the process. A second-order polynomial equation was employed, and ANOVA was applied to evaluate the impact of various operating parameters including extraction temperature(x_1; 44.9–70.1 °C), extraction time(x_2;5.0–10.0 h) and solvent to solid ratio(x_3;11.6–28.4 mL g^-1), on oil yield. Experiments to validate the model showed decent conformity between predicted and actual values. Extraction conditions for optimal oil yield were 61 °C, 8.75 h extraction duration and 19.25 mL g^-1 solvent to solid ratio. Under these conditions, the oil yield was predicted to be 5.1340%. Oil samples obtained were then analyzed using gas chromatography. The fatty acid composition revealed the major fatty acids to be oleic acid(C18:1) and linoleic acid(C18:2). The analysis of oil also demonstrated a decent ratio between omega-3 and omega-6 fatty acids. The structure of seeds was imaged using scanning electron microscopy. Oil quality was analyzed thermogravimetrically and by Fourier transform infrared spectroscopy. The assigned nutritional features of the D. lotus oil suggested that it can be used as an edible oil in pharmaceutical and food industry in the future.     

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non‐reproductive tissues

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.     

Effects of low‐density lipoproteins as additive on quality parameters and oxidative stress following cryopreservation of mithun (Bos frontalis) spermatozoa

Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p < .05) improvement in viability, sperm and nuclear abnormality, acrosome integrity, motility (progressive and in cervical mucus), cholesterol content, and reduction in the leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes was protected significantly (p < .05) in addition to significant (p < .05) improvement in binding per cent and binding index in IVF assay in extender containing 8% LDL. These results demonstrate that although cryopreservation of mithun's spermatozoa in EY was comparable with other species, addition of 8% LDL holds a clear advantage over EY or 10% LDL.     

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS. Methods:In the current investigation, a recombinant scFv mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results:Increased efficiency was achieved by EnBase® compared to LB broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC₅₀ for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion:Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv     

The effect of pH and calcium on copper availability to the springtail Folsomia candida in simplified soil solutions

The effect of pH and calcium on copper bioavailability to the springtail Folsomia candida was determined by assessing uptake kinetics upon copper exposure for 15 days in simplified soil solutions. A slight bioaccumulation of copper was observed in all treatments and controls. The effect of exposure concentration and calcium level on copper accumulation was not significant. Although pH and time slightly but significantly affected copper concentrations in the body, copper uptake rates were not significantly different from zero. This suggests that copper uptake in F. candida exposed to sub-lethal copper concentrations in soil pore water is not affected by pH and calcium.     

A review of thermal stress in cattle

Cattle control body temperature in a narrow range over varying climatic conditions. Endogenous body heat is generated by metabolism, digestion and activity. Radiation is the primary external source of heat transfer into the body of cattle. Cattle homeothermy uses behavioural and physiological controls to manage radiation, convection, conduction, and evaporative exchange of heat between the body and the environment, noting that evaporative mechanisms almost exclusively transfer body heat to the environment. Cattle control radiation by shade seeking (hot) and shelter (cold) and by huddling or standing further apart, noting there are intrinsic breed and age differences in radiative transfer potential. The temperature gradient between the skin and the external environment and wind speed (convection) determines heat transfer by these means. Cattle control these mechanisms by managing blood flow to the periphery (physiology), by shelter-seeking and standing/lying activity in the short term (behaviourally) and by modifying their coats and adjusting their metabolic rates in the longer term (acclimatisation). Evaporative heat loss in cattle is primarily from sweating, with some respiratory contribution, and is the primary mechanism for dissipating excess heat when environmental temperatures exceed skin temperature (~36°C). Cattle tend to be better adapted to cooler rather than hotter external conditions, with Bos indicus breeds more adapted to hotter conditions than Bos taurus. Management can minimise the risk of thermal stress by ensuring appropriate breeds of suitably acclimatised cattle, at appropriate stocking densities, fed appropriate diets (and water), and with access to suitable shelter and ventilation are better suited to their expected farm environment.