Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa

1. The effects of lycopene-enriched extenders on the in vitro quality of turkey semen including lipid peroxidation were examined after chilled and frozen storage.

2. Five pools of semen diluted in extenders containing 0, 0·05 or 0·1?mg/ml of lycopene were stored at 5°C for 48?h or cryopreserved as pellets and the following variables determined in fresh samples and samples stored chilled or frozen: sperm motility, viability, osmotic resistance, DNA integrity and lipid peroxidation (as malonaldehyde production).

3. Semen quality was generally compromised after storage, especially post-freezing. However, in the presence of the highest dose of lycopene, both the viability and osmotic-resistance of chilled spermatozoa and the DNA integrity of frozen spermatozoa were similar to those of fresh spermatozoa.

4. Greater lipid peroxidation was detected in refrigerated compared to fresh or cryopreserved spermatozoa. However, spermatozoa chilled in lycopene-enriched extenders showed significantly lower malonaldehyde levels than those chilled without lycopene, while the addition of lycopene to the freezing medium served to maintain the lipid peroxidation levels observed in fresh semen.

5. In conclusion, the presence of lycopene in the extender improved the survival of turkey spermatozoa after liquid-storage and protected DNA integrity against cryodamage. The beneficial effects of lycopene observed could be related to its capacity to diminish sperm lipid peroxidation during refrigeration or cryopreservation.     

Plumping fluid added to storage medium increases twofold the functional life span of fowl spermatozoa in vitro at 4°C

1. The objective of this study was to examine whether addition of plumping fluid (PF) to Lake's solution (LS) for storage of fowl spermatozoa in vitro at 4°C can prolong survival and improve the quality of spermatozoa.

2. In experiment 1, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 10%, 25%, 50% and 75% (v:v) PF for 0.5, 24, 48, 72, 96 and 120 h at 4°C. After the end of each storage period, spermatozoa were evaluated for their viability, mobility and penetrability. Viability was determined using SYBR-14 and propidium iodide (PI) staining. Mobility was assessed using an Accudenz assay. Penetrability was assessed using spermatozoa-inner perivitelline layer (IPL) interaction assay.

3. In experiment 2, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 25% and 50% (v:v) PF for 0.5, 24, 48 and 72 h at 4°C, and then fertility of the spermatozoa was evaluated using intravaginal artificial insemination (AI) in hens.

4. Storage of spermatozoa in LS alone resulted in loss of viability, mobility, penetrability and fertility within 48 h. In contrast, no loss of viability and penetrability was observed for the spermatozoa stored for 48, 96, 72 and 48 h in LS containing 10%, 25%, 50% and 75% (v:v) PF, respectively. In particular, fertilising capacity was not lost for the spermatozoa stored in the presence of 25% or 50% PF in LS for 48 and 24 h, respectively.

5. In conclusion, these findings demonstrated that in vitro exposure of fowl spermatozoa to PF during hypothermic storage in LS prolonged spermatozoa survival. A 25% (v:v) level of inclusion of PF in LS may be effective for the improvement of viability, penetrability and fertilising ability of the stored spermatozoa.     

Effect of different egg yolk-based extenders on the quality of ovine cauda epididymal spermatozoa during storage at 4°C

Cauda epididymal spermatozoa were obtained from testicles collected from abattoir(s). The pooled sperm samples were divided into four aliquots. Each aliquot was washed separately with the buffer of respective extender and finally extended with the four extenders viz. egg yolk–citrate (EYC), egg yolk–citrate–fructose (EYCF), Tris–citric acid–egg yolk–fructose (TCEYF) and egg yolk–Mcillvaine glucose (EYMG) and preserved at 4°C. The per cent sperm motility for EYC, EYCF, TCEYF and EYMG at 0 h was 50.83%, 56.67%, 75.00% and 31.67%, respectively, and at 72 h was 24.17% (EYC), 30.83% (EYCF), 51.67% (TCEYF) and 7.50% (EYMG). The corresponding figures for live sperm count at 0 h was 83.17%, 86.33%, 90.42% and 81.75% and at 72 h was 64.75%, 73.92%, 76.00% and 57.67%. The corresponding figures for mean per cent intact acrosome at 0 h was 95.33%, 95.50%, 90.92% and 97.25% and at 72 h was 86.17%, 83.92%, 77.58% and 86.33%. The sperm motility was significantly (p < 0.05) higher for TCEYF at different h of preservation from 0 h through 72 h. The sperm motility, live sperm count and per cent intact acrosome declined significantly (p < 0.05) with the advancement of storage time in all the four extenders. Our study concluded that TCEYF was best out of the extenders studied for preservation of cauda epididymal spermatozoa after double centrifugation and extension at 4°C up to 72 h of preservation. However, EYCF also has better potential for the preservation of cauda epididymal spermatozoa as viability was in close proximity and acrosomal integrity was higher compared with TCEYF extender.     

Identification and characterisation of microRNAs and Piwi-interacting RNAs in cockerels’ spermatozoa by Solexa sequencing

1. There has been substantial research focused on the roles of microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) derived from mammalian spermatozoa; however, comparatively little is known about the role of spermatozoa-derived miRNAs and piRNAs within breeding cockerels’ spermatozoa.

2. A small RNA library of cockerels’ spermatozoa was constructed using Illumina high-throughput sequencing technology. Unique sequences with lengths of 18–26 nucleotides were mapped to miRBase 21.0 and unique sequences with lengths of 25–37 nucleotides were mapped to a piRNA database. A total of 1311 miRNAs and 2448 potential piRNAs were identified. Based on stem-loop qRT-PCR, 8 miRNAs were validated.

3. Potential target genes of the abundant miRNAs were predicted, and further Kyoto Encyclopedia of Genes and Genomes database (KEGG) and Gene Ontology (GO) analyses were performed, which revealed that some candidate miRNAs were involved in the spermatogenesis process, spermatozoa epigenetic programming and further embryonic development.

5. GO and KEGG analyses based on mapping genes of expressed piRNAs were performed, which revealed that spermatozoal piRNAs could play important regulatory roles in embryonic development of offspring.

6. The search for endogenous spermatozoa miRNAs and piRNAs will contribute to a preliminary database for functional and molecular mechanistic studies in embryonic development and spermatozoa epigenetic programming.     

Changes in the acid‐base parameters of venous porcine blood caused by the period of storage and the method of sampling

Summary

The experiments demonstrate that the acid‐base status of porcine blood does not change significantly during a six hours’ storage period at 0° C. They further show that the sampling of blood via a vena cava puncture in comparison with sampling via an indwelling ear vein catheter is associated with severe stress that affects the acid‐base statuts of the blood.     

Effects of xylazine and dexmedetomidine on equine articular chondrocytes in vitro

Objective

To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro.

Effects of evisceration and storage temperature on changes in meat quality of traditional farm‐fresh Turkeys

1. Conditions affecting the keeping quality of traditional farm‐fresh turkeys were investigated.

2. Storage of uneviscerated Wrolstad turkeys at 4 °C for 10 days caused no statistically significant changes in meat flavour or texture.

3. During further storage at —2 °C, however, there was a slight but significant change in flavour, which became more marked with time in birds which had been eviscerated after the initial period at 4 °C.

4. Both eviscerated and uneviscerated birds became slightly tougher during storage.

5. Initial holding at 4 °C increased the numbers of psychrotrophic bacteria on the skin by about 10³ but subsequent changes at — 2 °C were slight for uneviscerated birds.

6. Eviscerated carcases had higher counts than uneviscerated birds after storage at — 2 °C and, although ‘off’ odours were not detected, spoilage appeared to be imminent at the end of the 20‐d period.     

The effect of tonicity of storage media for fowl semen on the occurrence of neck‐bending spermatozoa,fertility and hatchability 1

1. Semen of White Leghorn and Rhode Island Red males was stored for 24 h in diluents hypertonic (460 mOsm/kg H₂O) and isotonic (340 mOsm/kg H₂O) to cock seminal plasma.

2. Compared with the fertility results with semen that had been stored in the hypertonic diluent or was fresh, the fertility of the White Leghorns was not affected after storage in the isotonic diluent; a decrease (P < 0.lb05) was observed, however, using Rhode Island Red semen and isotonic diluent.

3. Fresh RIR semen contained 2.lb31% “neck‐bent spermatozoa” (NBS) which was increased to 4.lb23% and 5.lb76% after dilution in hypertonic and isotonic diluents respectively and stored for 24 h. It is doubtful whether this increase (P < 0.lb05) is the sole reason for the lowered fertility obtained with this breed after storage in the isotonic diluent.     

The role of Bcl-xL and nuclear factor-��B in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-_L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.     

Role of interleukin-1β in the regulation of porcine corpora lutea during the late luteal phase of the cycle and during pregnancy

Interleukin-1β (IL-1β) may regulate ovarian physiology. In this study, the influence of IL-1β on secretory activity within the corpora lutea (CL) of cyclic and gravid pigs was determined in vitro during different stages of the CL lifespan, e.g. on Days 10-11, 12-13 and 15-16 of the oestrous cycle and pregnancy. IL-1β (10 ng/ml) increased prostaglandin E2 (PGE2) secretion from CL of the cyclic and gravid pigs during studied days of the oestrous cycle and pregnancy. Increase (P < 0.05) of prostaglandin F2α (PGF2α) in IL-1β-treated CL was demonstrated only on Days 10-11 of the oestrous cycle. More potent stimulatory effect of IL-1β on PGE2 than PGF2α secretion resulted in the enhancement of the PGE2:PGF2α ratio in cyclic and early pregnant CL. IL-1β increased (P < 0.05) progesterone (P4) secretion only in gravid CL and had no effect on oestradiol-17β (E2) release. Expression of cyclooxygenase-2 (COX-2) mRNA was stimulated (P < 0.05) in IL-1β-treated cyclic and gravid CL. Expression of prostaglandin synthase mRNAs in response to IL-1β did not increase. In conclusion, IL-1β modulates PGE2, PGF2α and P4 secretion from porcine CL, depending on luteal stage and the surrounding hormonal milieu. The cytokine may act locally in porcine CL for luteotrophic support throughout the PGE2-mediated synthesis and secretion.     

Oxidative stability and α‐tocopherol retention in turkey burgers during refrigerated and frozen storage as influenced by dietary α‐tocopheryl acetate

1. The effect of vitamin E (α‐tocopheryl acetate) in turkey diets on the oxidative stability of raw and cooked turkey burgers and on the retention of α‐tocopherol during refrigerated (4°C) or frozen (‐20°C) storage was investigated. One hundred and two, one‐day‐old T‐8^S turkey poults were divided at random into 3 groups of 34 animals each and fed on either a basal diet (normal commercial turkey diet) supplemented with 20 mg α‐tocopheryl acetate/kg (control) or fed an α‐tocopherol supplemented diet containing 300 (E300) or 600 (E600) mg α‐tocopheryl acetate/kg for 21 weeks.

2. Dietary supplementation with α‐tocopheryl acetate significantly reduced TBARS numbers in both raw and cooked burgers during refrigerated and frozen storage.

3. The mean values of α‐tocopherol in raw and cooked burgers stored at 4°C did not change during storage.

4. In the case of both raw and cooked samples stored at ‐20°C, the α‐tocopherol values decreased from 5.67 to 3.54 and from 3.56 to 2.30 μg/g in the raw burgers from turkeys from the E600 and E300 treatments, respectively, after 4 months storage. The values decreased from 5.60 to 2.88 and from 3.29 to 1.85 μg/g in cooked burgers from turkeys from the E600 and E300 treatments, respectively, after 5 months storage.     

Variation among individual white‐leghorn hens in the concentration of minerals in the albumen and yolk content of their eggs

1. The mineral composition of the albumen and yolk was determined in several eggs from each of a number of individual hens from the same White‐Leghorn strain. X‐ray fluorescent spectrometry was used to undertake two independent series of analyses. A total of 8 minerals (calcium, chlorine, iron, magnesium, phosphorus, potassium, sodium, sulphur) were included in the analyses of the yolk and the same minerals, but excluding iron (which is present in only small amounts), in the albumen.

2. There was considerable variation between individuals in the mineral concentration in their eggs (coefficients of variation ranged between 3.8% for sodium to 19.9% for calcium in the albumen, and between 4.3% for phosphorus to 11.8% for iron in the yolk).

3. At the same time, the moderately high repeatability of mineral concentration (t = 0.4?0.6) in successive eggs from the the same hen for several of the minerals analysed is indicative of some positive control by the hen of the mineral composition of her eggs.

4. There was a highly significant correlation (P≤ 0.001) between the mean concentration of potassium in the albumen and the hatchability of the eggs, supporting the claim that a deficiency of potassium in the egg could be the basis of some failures in hatchability.

5. The study also revealed variation among individual birds in the concentration of iron in the yolk which was negatively correlated (P≤0.01) with hatchability. No clear basis could be suggested for this variation among individual birds.     

Structural and molecular pathology of the atrium in boxer arrhythmogenic right ventricular cardiomyopathy

Objective

To investigate the expression and distribution of desmosomal and gap junction proteins of the intercalated disc in the atria of boxers with arrhythmogenic right ventricular cardiomyopathy (ARVC).

Alterations in vaginal temperature during the estrous cycle in dairy cows detected by a new intravaginal device—a pilot study

Tropical Animal Health and Production - Estrus identification is important in dairy cow production. At present, estrus identification is automated with a pedometer or accelerometer and the results...     

Fluorescein‐diacetate,a fluorescent dye compound stain for rapid evaluation of the viability of mammalian oocytes prior to in vitro studies

Summary

A method for a quantitative measurement of the fluorescence activity of porcine egg cells is described. The non‐polar fluorescein‐diacetate molecules enter the cell, are hydrolyzed by cell esterases, and fluorescein is produced. This polar compound can not leave the cell because it is unable to pass through the intact cell membrane, and it therefore accumulates in the cytoplasm of the cell. Damaged cells however show a distinct loss of fluorescein through the cell membrane. With the aid of a fluorescence microscope, a photometer and a recorder, the amount of radiated light can be measured. The advantages of this method in oocyte research are briefly discussed.     

Relationship between contractions of the uterus and concentration of PGF2α in uterine venous blood after luteolysis in gilts

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.     

Involvement of tannins and flavonoids in the in vitro effects of Newbouldia laevis and Zanthoxylum zanthoxyloïdes extracts on the exsheathment of third-stage infective larvae of gastrointestinal nematodes

The present study aimed at examining the possible role of tannins and flavonoids on the in vitro anthelmintic properties of the extracts of two plants from the southern area of Western Africa, i.e. Newbouldia laevis and Zanthoxylum zanthoxylo?des. Extracts of the two plants were prepared by use of acetone/water (70/30) and their anthelmintic activity was measured by use of the larval exsheathment inhibition assay (LEIA) applied on the abomasal species, Haemonchus contortus and the intestinal species Trichostrongylus colubriformis. Three concentrations of extracts were evaluated to examine the possible dose effect. In addition, the possible involvement of tannins and flavonoids was examined by comparing the levels of inhibition of larval exsheathment obtained with the same extracts, after of not addition of PVPP which forms complexes with these compounds. The results indicate significant effects with both plants and both nematode species. In the range of concentrations examined, the results were dose-dependent for N. laevis extracts but not for Z. zanthoxylo?des because the three doses applied provoked a similar highly significant inhibition whatever the tested dose. The use of PVPP indicated for both plant and nematode species, that tannins and flavonoids are involved partly in the effect but that some other biochemical compounds were also involved in both plants.     

Expression kinetics of chicken β2-microglobulin and Class I MHC in vitro and in vivo during Marek’s disease viral infections

Marek’s disease virus (MDV) is a highly cell-associated herpesvirus that causes a disease in chickens characterized by tumor formation and immunosuppression. The changes of major histocompatibility complex (MHC) expression in different MDV-infected cells are not completely understood. In this study, we investigated the expression of the Class I MHC and β2-microglobulin (β2m) genes in response to MDV infection at different time points by real-time PCR. In both in vitro and in vivo, the expression levels of Class I MHC and β2m genes were upregulated during early MDV infections in comparison to control cells; We also found that the expression of Class I MHC gene was downregulated in BudR (5-bromo-2′-deoxyuridine)-treated MSB1 cells at 48 h and MDV-infected chicken embryo fibroblast cells (CEF) at 120 and 168 h post infection (hpi); Furthermore, compared to control groups, Class I MHC and β2m expression levels were downregulated in peripheral blood lymphocytes (PBLC) from MDV-infected chickens at 14 and 28 days post infection (dpi); Interestingly, both Class I MHC and β2m gene expression levels increased again in PBLC from MDV RB1B-infected chickens at 35 dpi, in which MDV was in the latent or transformed infection stages. In addition, Class I MHC expression was clearly decreased in MDV-infected CEF at 120 hpi although β2m expression was significantly increased. These changes in Class I MHC and β2m gene expression might provide more insights into host-virus interaction.