Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30-90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = -0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm-oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.     

Freeze-dried turkey muscle I.—Changes in nitrogenous compounds and lipids of dehydrated turkey during storage

Changes in proteins and lipids were followed during the storage of freeze-dried turkey breast muscle in air and in nitrogen. Oxidation of sulphydryl groups accounted for part of the oxygen uptake of air-stored samples, and was accompanied by a decrease in soluble nitrogen greater than in controls stored in nitrogen. The major deteriorative process at low moisture content was a type of ‘lipid’ browning reaction which was dependent on oxygen and caused discoloration and objectionable odours. Autoxidation of lipids catalysed by haem pigments and resulting in rancid flavours was not a major deteriorative process in freeze-dried turkey muscle. Lipid hydrolysis occurred at water contents of 8·2 and 5·4% but not at 0·8%.     

Long-term storage of mouse spermatozoa after evaporative drying

To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.     

Oxidative and sensory changes during bulk and retail storage of hot-filled turkey casserole

 Oxidative and sensory changes in hot-filled turkey casserole were investigated during bulk storage at 3°C and during retail storage at 4°C. Oxidative changes, assessed as warmed-over flavour (WOF) by a trained sensory panel or determined as levels of thiobarbituric acid reactive substances (TBARS), showed no development during bulk storage for up to 3 weeks, whereas scores of WOF and levels of TBARS increased rapidly during retail storage for up to 3 days. The development of WOF during retail storage was avoided when the product was vacuum-packed or gas-packed. Only insignificant microbiological changes were observed during bulk storage of the product or during the subsequent retail storage. Received: 8 October 1997 / Revised version: 19 December 1997     

Effect of irradiation on the quality of turkey ham during storage

Effect of electron-beam irradiation on the quality of ready-to-eat (RTE) turkey ham was studied. Turkey hams were purchased from local stores and sliced into 0.5 cm-thick pieces and vacuum packaged. The ham samples were randomly separated into three groups and irradiated at 0, 1, or 2 kGy, and stored at 4?°C for up to 14 days. Volatiles, color, TBARS values and sensory characteristics were determined to compare the effect of irradiation and storage on the quality of RTE turkey ham. Irradiation had little effects on color and TBARS values of RTE turkey hams. Sensory analysis indicated that sulfury odor increased as irradiation dose increased, and the contents of sulfur compounds in irradiated RTE turkey hams were higher (P <0.05) than those in nonirradiated samples. Irradiation increased (P <0.05) the production of acetaldehyde, which could be related to a metal-like flavor in irradiated hams. However, overall quality changes in RTE turkey hams by irradiation up to 2 kGy were minor.     

Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

At the avian body temperature of 40 degrees C, intact fowl spermatozoa require Ca(2+) for the initiation of motility and a combination of both Ca(2+) and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1-100 micromol/l, neither PD 150606 (a Ca(2+)-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca(2+)-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca(2+) and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca(2+), as well as motility initiated by calyculin A -- a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 degrees C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.     

Induced hyperactivity in boar spermatozoa and its evaluation by computer-assisted sperm analysis

Hyperactivity, a form of sperm motility characterized by vigorous flagellar movements, has been proposed as essential for fertilization in mammals. The objective of the present study was to establish a method for inducing hyperactivity in vitro in boar spermatozoa and to define threshold values to differentiate between hyperactive and non-hyperactive spermatozoa by computer-assisted sperm analysis (CASA) as a prerequisite for analyzing the energy metabolism during hyperactivity. In TALP-HEPES medium, non-frozen boar spermatozoa were stimulated to hyperactivity by 50 micromol l(-1) Ca2+ within 15 min at 37 degrees C if 5 micromol l(-1) of the Ca2+ ionophore A23187 was present. If 25% seminal plasma was present, boar spermatozoa required higher Ca2+ concentrations (about 700 micromol l(-1)) for hyperactivity. Under both conditions, immobilization and head-to-head agglutination were low so that hyperactive spermatozoa could be analyzed for at least 40 min. The transition from normal to hyperactive movement was characterized by an increase in flagellar beat angle from 49 degrees +/- 12 degrees to 200 degrees +/- 36 degrees (n = 32) and a decrease in flagellar curvature ratio from 0.89 +/- 0.04 to 0.47 +/- 0.11 (n = 32). For quantification of hyperactive boar sperm, kinematic parameters of hyperactive and non-hyperactive spermatozoa were measured by CASA and statistically evaluated (receiver operating characteristic (ROC) curve analysis). The threshold values of the following four parameters were well suited for differentiating between hyperactive and non-hyperactive boar spermatozoa (ROC curve analysis: > 50% specificity at 100% sensitivity). Hyperactive boar spermatozoa showed mean lateral head displacement > 3.5 microm, curvilinear velocity > 97 microm s(-1), linearity < 32% and wobble < 71%. According to this multiparametric definition, induction of hyperactivity increased significantly (P < 0.0001) the fraction of hyperactive spermatozoa in semen samples from 5.1 +/- 4.3% (n = 13) to 48.3 +/- 6.6% (n = 7) in the absence and to 44.2 +/- 7.6% (n = 10) in the presence of 25% seminal plasma, while the overall percentage of motile spermatozoa did not change significantly.     

Changes in the lipids of turkey muscle during storage at chilling and freezing temperatures

Diced turkey leg and breast muscle stored for 22 days at 0° and —3°, and for up to one year at —10°, —20° and —60° was examined at intervals for lipid composition. Free fatty acids increased at all temperatures except —60°, with a Q₁₀ of 3-4 between 0° and —20° 90% of the fatty acids liberated were unsaturated, matching in composition the unsaturated acids of the muscle glycerophospholipids. Demonstration of linear relationships between increase in free fatty acids and decrease in phosphatidylethanolamine or increase in lysophosphatidylcholine confirmed phospholipase A₂ as the enzyme mainly responsible. The composition of the fatty acids liberated during storage at 0° and —3° showed that both lipase and phospholipase were active. A slight decrease in extractability, resulting in an apparent loss of phospholipid-P, was observed after storage at low temperatures.     

Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility

Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osm(crit)) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 degrees C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osm(crit) at 15 degrees C was 54.7+/-3.2, 68.5+/-10.6, 116.7+/-2.1 and 194.3+/-3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osm(crit)=67.8+/-2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.     

Effect of high-pressure treatment on lipid oxidation in turkey thigh muscle during chill storage

 Formation of secondary lipid oxidation products during chill storage of vacuum-packed (99% vacuum), pressure-treated turkey thigh muscle was found to depend on working pressure (pressure range up to 500 MPa at 10°C) and to a lesser degree on pressurization time (10 and 30 min). Pressure treatment at 400 MPa and lower pressures for 30 min (and for 10 min) resulted in less formation of thiobarbituric-acid-reactive substances (TBARS) during 6 days of storage at 5°C compared to heat treatment at 100°C for 10 min, while pressure treatment at 500 MPa for 30 min gave similar development of TBARS as did the heat treatment. The formation of TBARS during storage at 5°C was found to depend exponentially on the pressure used for treatment at both 10 min and 30 min, and apparent volumes of activation are proposed as a parameter for quantification of the effects of pressure on lipid oxidation in meat during subsequent storage. Received: 18 November 1996     

Effects of nitrite levels,endpoint temperature and storage on pink color development in turkey rolls

The objective of this research was to determine the relationship between minimum nitrite level, endpoint temperature and storage period on pink discoloration of turkey rolls. Rolls were prepared with 0, 3 or 5 parts per million (ppm) NO₂^–, cooked to an endpoint temperature of 75 °C or 85 °C and stored at 4 °C. During the 13-day storage period, on the 0th, 4th, 8th and 13th days cured pigment, total pigment, residual nitrite and color parameters were measured and sensory color was evaluated. At each endpoint temperature, addition of 3 ppm or 5 ppm NO₂^– significantly increased cured pigment and total pigment levels. Cured pigment levels of turkey rolls cooked to 75 °C were higher than rolls cooked to 85 °C, regardless of the NO₂^– level. There was a significant decrease in cured pigment levels at the end of the storage period (13th day). Analysis of residual NO₂^– in all treatment groups showed no detectable amounts. Endpoint temperature and addition of NO₂^– affected L* values: rolls cooked to the 75 °C endpoint had higher L* values. The additon of nitrite with the 75 °C endpoint temperature resulted in higher a* values. a* values also increased during the storage. The higher endpoint temperature (85 °C) resulted in lower a* values. Yellowness was not affected by final cooking temperature. Both the addition of NO₂^– and storage decreased b* values. The panel found no differences in pinkness intensity between the two levels of added nitrite (3 and 5 ppm). Sensory pinkness intensity in nitrite-added samples increased with increasing storage.     

Sperm surface changes and physiological consequences induced by sperm handling and storage

Spermatozoa interact with their immediate environment and this contact remodels the sperm surface in preparation for fertilisation. These fundamental membrane changes will be critically covered in this review with special emphasis on the very specific surface destabilisation event, capacitation. This process involves very subtle and intricate modifications of the sperm membrane including removal of suppression (decapacitation) factors and changes in the lateral organisation of the proteins and lipids of the sperm surface. Processing of sperm for assisted reproduction (storage, sex-sorting, etc.) subjects spermatozoa to numerous stressors, and it is possible that this processing overrides such delicate processes resulting in sperm instability and cell damage. To improve sperm quality, novel mechanisms must be used to stabilise the sperm surface during handling. In this review, different types of membrane stress are considered, as well as novel surface manipulation methods to improve sperm stability.     

Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.     

Effect of packaging and storage time on survival of Listeria monocytogenes on kippered beef steak and turkey tenders

The objective of our study was to determine effect of packaging method and storage time on reducing Listeria monocytogenes in shelf-stable meat snacks. Commercially available kippered beef steak strips and turkey tenders were dipped into a 5-strain L. monocytogenes cocktail, and dried at 23 °C until a water activity of 0.80 was achieved. Inoculated samples were packaged with 4 treatments: (1) vacuum, (2) nitrogen flushed with oxygen scavenger, (3) heat sealed with oxygen scavenger, and (4) heat sealed without oxygen scavenger. Samples were stored at 23 °C and evaluated for L. monocytogenes levels at 0, 24, 48, and 72 h. Initial levels (time 0) of L. monocytogenes were approximately 5.7 log CFU/cm2 for steak and tenders. After 24 h of storage time, a 1 log CFU/cm2 reduction of L. monocytogenes was observed for turkey tenders for all packaging treatments. After 48 h, turkey tenders showed >1 log CFU/cm2 reduction of L. monocytogenes for all packaging treatments except for vacuum, where only 0.9 log CFU/cm2 reduction was observed. After 72 h, reductions for all packaging treatments for turkey tenders ranged from 1.5 to 2.4 log CFU/cm2. For kippered beef steak, there was no interaction between the packaging treatments and all storage times (P > 0.05) whereas, time was different (P <0.05). For kippered beef steak, there was 1 log reduction of L. monocytogenes at 24 and 48 h of storage times at 23 °C for all packaging treatments and a 2.1 log CFU/ cm2 L. monocytogenes reduction at 72 h of storage time. PRACTICAL APPLICATIONS: Processors of kippered beef steak and turkey tenders could use a combination of vacuum or nitrogen-flushing or heat sealed with an oxygen scavenger packaging methods and a holding time of 24 h prior to shipping to reduce potential L. monocytogenes numbers by ≥1 log. However, processors should be encouraged to hold packaged product a minimum of 72 h to enhance the margin of safety for L. monocytogenes control.     

Evolution of amino acids and biogenic amines throughout storage in sausages made of horse,beef and turkey meats

The changes in concentration of free amino acids and biogenic amines, along 28 d of storage at 4 °C, were monitored in a wide range of European ripened sausages manufactured from horse, beef and turkey meats. Generally speaking, both chemical families became more concentrated with elapsing time – but rather distinct patterns were followed in each meat type: total free amino acids increased by 13-fold in the case of horse sausages, and 5-fold in the case of beef sausages, but decreased to one third in the case of turkey sausages; and total biogenic amines attained 730 mg/kg in turkey sausages, 500 mg/kg in beef sausages and 130 mg/kg in horse sausages by 28 d of refrigerated storage. For putrescine, maximum levels of 285 mg/kg were attained in turkey and 278 mg/kg in beef sausages; for cadaverine, maximum levels of 6 mg/kg in turkey and 9 mg/kg in beef; and for histamine, maximum levels of 263 mg/kg in turkey and 26 mg/kg in beef. Hence, public safety concerns may be raised in the case of turkey sausages.     

Development of warmed-over flavour in ground turkey, chicken and pork meat during chill storage. A model of the effects of heating temperature and storage time

The susceptibility towards development of warmed-over flavour (WOF) was investigated in meat from turkey and chicken breast and thigh, and from pork longissimus dorsi muscle. Ground meat samples from these five sources were heated for 30 min in a water bath at 60, 70 or 80 °C, and the samples were stored at 5 °C for 0–4 days. During storage, WOF was quantified by measurement of thiobarbituric-acid-reactive substances (TBARS) and by sensory evaluations. The increase in TBARS was modelled for each type of meat at the different heating temperatures by a first-order reaction, and it was shown that a common rate constant could be used for all types of meat. The estimated maximum levels of TBARS in meat samples decreased in the following order: turkey thigh > chicken thigh > turkey breast > chicken breast > pork. For each type of meat, the estimated maximum level of TBARS rose when the heating temperature increased in the range 60–80 °C. This temperature effect was particularly obvious for the chicken samples. Thus thigh and breast meat from chicken heated to 60 °C was almost stable against oxidation during storage. Results obtained by measurement of TBARS were in good agreement with the sensory evaluations.

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Entwicklung von Aufwärmegeschmack in gehacktem Fleisch von Pute, Hähnchen und Schwein während der Kühllagerung. Ein Modell des Effektes der Aufwärmetemperatur und Lagerungzeit

Zusammenfassung Die Entwicklung des Aufwärmegeschmacks (WOF) in Brust und Schenkel von Pute und Hähnchen und im Longissimus dorsi vom Schwein wurde untersucht. Fünf Sorten von gehackten Fleischproben wurden in einem Wasserbad bei 60, 70 oder 80 °C aufgewärmt und 0–4 Tage bei 5 °C aufbewahrt. In der Lagerungzeit wurde WOF über Thiobarbitursäure-reaktive Substanzen (TBARS) und sensorische Beurteilungen bestimmt. Die Erhöhung der Mengen von TBARS für eine Fleischsorte bei einer gegebenen Temperatur wurde mit einer mathematischen Funktion 1. Ordnung beschrieben, und eine gemeinsame Reaktionskonstante für alle Fleischsorten gefunden. Die bestimmten Maximumwerte für TBARS waren in fallender Ordnung: Putenschenkel > Hähnchenschenkel > Putenbrust > Hähnchenbrust > Schwein. Für eine gegebene Fleischsorte stiegen die gemessenen Maximumwerte für TBARS mit erhöhten Aufwärmetemperaturen im Bereich von 60–80 °C. Dieser Temperatureffekt war im besonderen für Hähnchenfleisch anzumerken und äußerte sich bei fast unveränderter Oxidationsstabilität in Schenkel und Brustfleisch von Hähnchen, die bei 60 °C aufgewärmt wurden. Die bei den TBARS bestimmten Resultate waren in guter Übereinstimmung mit den sensorischen Beurteilungen.

     

鱼精蛋白对延长鱼糕制品有效保存期的作用

本文用鱼精蛋白、苯甲酸钠和山梨酸钾分别添加在鱼糕制品中，并于12℃和24℃条件下保存，观察其保存效果。结果表明：添加0．8％鱼精蛋白的鱼糕在12℃和24℃的有效保存期分别为7d和5d，达到添加0．3％苯甲酸钠和0．2％山梨酸钾的效果。