One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles.

Milk extracellular vesicles (EV) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EV with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2 quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EV size, concentration, or morphology. We did not detect any changes in the EV cargo regarding the amount of protein and RNA, nor in the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low SCC. Overall, we observed fewer CD81 and CD9 markers in quarters with high SCC. Moreover, we found no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to 1 wk is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EV are highly stable in the raw milk matrix.

Effects of time and temperature of storage on chemical and nutritional characteristics of raw milk for Provolone Valpadana PDO cheesemaking: a multivariate approach.

We evaluated the possibility of increasing the storage temperature of raw milk for Provolone Valpadana cheesemaking, to identify the most suitable conditions of time and temperature for a pre-maturation process. We used Principal Component Analysis (PCA) to analyze the overall effects of different storage conditions on chemical, nutritional and technological characteristics of the raw milk. Four different thermal storage cycles, two at fixed temperature/time (6 and 12°C for 60 h) and two with two-phase thermal cycle (10 and 12°C for 15 h, followed by refrigeration at 4°C for 45 h) were studied. Although a moderate heterogeneity among raw milks from the 11 producers of Provolone Valpadana cheese was observed, PCA revealed the critical aspects of the extreme storage conditions (60 h of refrigeration). Some samples resulted in anomalous behaviors, probably related to unexpected fermentation phenomena occurring with increasing storage temperature. The acidification and the increase in the contents of lactic acid, soluble calcium, and degree of retinol isomerization observed in the anomalous samples can compromise the technological functionality of milk. Conversely, the storage with a two-phase thermal cycle did not lead to variations in any measured characteristic, suggesting that mild refrigeration conditions (10 or 12°C for 15 h followed by 4°C for 45 h) could be a good compromise in favoring milk pre-maturation without altering its quality characteristics.

Screening of frozen-thawed conditions for keeping nutritive compositions and physicochemical characteristics of goat milk.

Frozen milk can help producers overcome the seasonality of goat milk production, low goat production and short lactation periods, and avoid discarding milk during some special periods. We investigated effects of combination between freezing (cryogenic refrigerator of -16 to -20°C or ultra-cryogenic refrigerator of -76 to -80°C) and thawing (homeothermy of 20 to 25°C or refrigeration of 2 to 4°C) on nutritive compositions and physicochemical characteristics of raw goat milk during storage period (80 d). Compared with fresh goat milk, the frozen-thawed milk decreased contents of fat, protein, and lactose, as well as surface tension and stability coefficient, whereas increased effective diameter and polydispersity index. The average values of color values (L*, a*, and b*) in 4 group samples changed from 83.01 to 82.25, -1.40 to -1.54, 3.51 to 3.81, respectively, and the ΔE of most samples did not exceed 2. In contrast to the other 3 frozen-thawed treatments, goat milk treated with ultra-cryogenic freezing-homeothermic thawing (UFHT) possessed higher fat (5.20 g/100 g), smaller effective particle diameter (0.32 µm), and the lowest polydispersity index value (0.26). The color and confocal laser scanning microscopy images of UFHT were similar to those of fresh goat milk, illustrating UFHT was the optimal approach to maintain the natural quality of goat milk. Our finding provides a theoretical basis for producers to freeze surplus milk.

Technical note: Effect of delayed analysis of cooled lithium-heparinized whole blood on the stability of ionized calcium, ionized magnesium, sodium, potassium, chloride, glucose, and lactate in samples from dairy cows.

The objectives of this study were to describe the stability of bovine whole-blood electrolytes, glucose, and lactate in samples collected in lithium heparin tubes and stored in thermoconductive modules immersed in ice water. A total of 99 Jersey cows (40 first-parity, 18 second-parity, and 41 third-parity or greater cows) from a commercial dairy farm in West Texas were enrolled between June and July 2018. Blood was collected from the jugular vein using a 60-mL polypropylene syringe and equally distributed into 5 spray-dried evacuated lithium heparin tubes. Baseline samples were analyzed within 90 s of collection using a benchtop blood gas analyzer. The remaining 4 tubes were stored in a thermoconductive, passive-temperature-regulating module inside a cooler with ice water. At 30 min and 2, 4, and 8 h post-collection, samples were removed from the temperature-regulating module, gently inverted for 10 s, and analyzed. Repeated-measures models were built to evaluate the effect of time on the stability of ionized Ca (iCa), ionized Mg (iMg), Na, K, Cl, glucose, and lactate. Most of the analytes investigated remained stable up to 8 h under ice water storage conditions before analysis, including iCa, iMg, Cl, glucose, and lactate. However, Na and K were significantly affected by delayed analysis: Na remained stable up to 4 h post-collection, but K was not stable starting at 2 h post-collection. The results of this study are useful in helping future researchers and consultants to recognize acceptable time delays between whole blood collection and processing or analysis for electrolytes, glucose, and lactate.

On-farm storage of livestock vaccines may be a risk to vaccine efficacy: a study of the performance of on-farm refrigerators to maintain the correct storage temperature.

BACKGROUND: Livestock vaccines (LV) are often stored on-farm, in a refrigerator (fridge), prior to use and little is documented about the storage conditions during this period. As the quality of a vaccine can be impaired by storage at an incorrect temperature, the present study aimed to evaluate the on-farm performance of farm fridges to maintain the correct storage temperature. From January to August 2014, temperature data loggers were placed on selected farms fridges used to store LV (n = 20) in South-West England. RESULTS: Temperature recording data was available from 17 of the 20 farms. Fifty-nine percent of farm fridges had at least one temperature recording above 8 °C, 53% had at least one recording below 2 °C and 41% at or below 0 °C. Internal fridge temperatures attained 24 °C and dropped to - 12 °C as an absolute maximum and minimum respectively. Fridges tested spent an average of 16% of the total time recorded above 8 °C. Time of the year significantly influenced the percentage of time above 8 °C. External and internal temperatures were found to be positively correlated (p < 0.001). Statistical significant differences in internal and external temperatures were found between March and August. CONCLUSIONS: The majority of fridges in this study would have failed to keep any stored LV within the recommended storage temperature range. If LV are going to be stored on-farm prior to use, then urgent improvements in this part of the cold-chain are required in order to insure vaccine efficacy is not compromised.

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa.

Metformin is clinically used to treat diabetes. Given its role-impacting metabolism, metformin has been also added to semen cryopreservation media showing specie-dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin-including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ-population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.

Influence of Post-Mortem Sperm Recovery Method and Extender on Unstored and Refrigerated Rooster Sperm Variables.

Many post-mortem sperm collection techniques have been described for mammalian species, but their use in birds is scarce. This paper compares the efficacy of two post-mortem sperm retrieval techniques - the flushing and float-out methods - in the collection of rooster sperm, in conjunction with the use of two extenders, i.e., L&R-84 medium and Lake 7.1 medium. To determine whether the protective effects of these extenders against refrigeration are different for post-mortem and ejaculated sperm, pooled ejaculated samples (procured via the massage technique) were also diluted in the above extenders. Post-mortem and ejaculated sperm variables were assessed immediately at room temperature (0 h), and after refrigeration at 5°C for 24 and 48 h. The flushing method retrieved more sperm than the float-out method (596.5 ± 75.4 million sperm vs 341.0 ± 87.6 million sperm; p < 0.05); indeed, the number retrieved by the former method was similar to that obtained by massage-induced ejaculation (630.3 ± 78.2 million sperm). For sperm collected by all methods, the L&R-84 medium provided an advantage in terms of sperm motility variables at 0 h. In the refrigerated sperm samples, however, the Lake 7.1 medium was associated with higher percentages of viable sperm, and had a greater protective effect (p < 0.05) with respect to most motility variables. In conclusion, the flushing method is recommended for collecting sperm from dead birds. If this sperm needs to be refrigerated at 5°C until analysis, Lake 7.1 medium is recommended as an extender.

Storage of gastrointestinal nematode infective larvae for species preservation and experimental infections.

Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes are of considerable interest to preserve rare species and to maintain a stable source for routine experimental infections. This study compares the relative pros and cons of the two most common techniques, cryopreservation and refrigeration by comparing how they influence consequent infection outcome parameters in terms of life-history traits and fitness as a function of time using the gastrointestinal nematode of sheep Haemonchus contortus as a study species. Establishment capacity was found to be significantly reduced in cryopreserved stocks of L3 compared to refrigerated stocks, but this was followed by significant increases in their fecundity. Refrigeration did not affect L3 stocks consequent fitness by 16 months (the maximum examined) although they did incur a significant reduction in establishment, followed once again by an augmentation in fecundity. The study highlights potential areas for bias in comparing studies using L3 larvae maintained for different periods of time under different techniques.

Effect of Conjugated Linoleic Acid on Boar Semen Quality After Long-term Refrigeration at 17°C.

In this study, the effect of conjugated linoleic acid (10 trans, 12 cis) (CLA) on refrigerated boar sperm quality parameters up to 14 days at 17°C was assessed. Semen was extended in Androhep and divided into four treatments supplemented with CLA (25, 50, 100 and 200 µm) and control group, then kept for 2 h at 22°C. Afterwards an aliquot of each treatment was removed, and mitochondrial activity, viability, lipid membrane peroxidation (LPO) and stability of the sperm plasma membrane were assessed by flow cytometry. The remaining extended semen was maintained at 17°C until 336 h, repeating the same analysis every 48 h. Regarding percentage of live spermatozoa, no statistical differences were observed among treatments up to 96 h. After this time, viability decreased significantly (p < 0.05) for CLA concentrations of 100 and 200 µm. Despite these results, there was an individual response to CLA. Although in the control group, the boar A presented better results when compared with the other boars, especially at concentrations of 50 and 100 µm boar B showed significantly higher results (p < 0.05). Supplementation with CLA improved (p < 0.05) LPO, but not the mitochondrial membrane potential of sperm. The highest two CLA concentrations showed to be toxic for sperm as all results were lower than the observed for the control. In conclusion, CLA at 50 µm seems to be an efficient concentration for reducing the oxidative stress, decreasing LPO, maintaining viability, membrane stability and mitochondrial potential on refrigerated boar spermatozoa.

Comparison between commercial media and TRIS-egg yolk extender in the refrigerated storage of collared peccary semen at 17°C.

To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48 hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48 hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48 h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around ∼30-50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48 hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and ∼ 60-70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48 hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose.

Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage.

Anurans exhibit a greater reproductive diversity than any other vertebrate order. However, studies investigating the effects of the external fertilisation environment on fertilisation success are limited to aquatic-breeding species. This study investigated the effects of fertilisation medium osmolality, sperm concentration and short-term oocyte storage on fertilisation success in a terrestrial-breeding anuran, Pseudophryne guentheri. Split-clutch experimental designs were used to determine optimal fertilisation conditions. To determine the effect of short-term sperm storage, sperm viability was assessed using fluorescence microscopy and percentage sperm motility and velocity quantified with a computer-assisted sperm analysis system. Fertilisation success was highest in media ranging in osmolality from 25 mOsm kg⁻¹ to 100 mOsm kg⁻¹, representing a broader range and higher optimal osmolality than previously reported for aquatic breeders. High rates of fertilisation (>75%) were achieved in relatively low sperm concentrations (2.5×104 mL⁻¹). Oocytes stored in isotonic solutions (200 mOsm kg⁻¹) retained fertilisation capacity (32%) after 8h of storage, while sperm suspensions maintained motility (≥26%) for 13 days. Additional studies on terrestrial-breeding anurans will be required to ascertain whether the optimal fertilisation conditions reported reflect adaptations to achieve fertilisation in a terrestrial environment.

Applicability of a new cell culture device for cooled-storage of stallion semen.

A new device for storage and shipping of cell cultures--the Petaka G3 cell management device--was tested for its applicability for cooled-storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg/l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15 °C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer-assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15 °C than at 5 °C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled-storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.

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.

Boar variability affects sperm metabolism activity in liquid stored semen at 5 degrees C.

Metabolic activity of boar spermatozoa, liquid stored for three days at 5 degrees C, was measured using bioluminescence for ATP content, fluorescent assay (JC fluorochrome) of mitochondrial activity and oxygen consumption. Sperm motility and plasma membrane integrity (PMI) were simultaneously analyzed. Apart from the statistically significant effect (P < 0.001) of semen storage time, the importance of the individual source of the ejaculate for the analyzed parameters of metabolic efficiency of spermatozoa was shown. This phenomenon was manifested in the interaction of the individual source of the ejaculate with spermatozoa motility, integrity of their membranes and metabolic activity with the passing time of semen preservation. Recorded results indicate that the individual factor may have a significant influence on the technological usefulness of boar spermatozoa for liquid storage. Quality analyses conducted on boar semen stored at 5 degrees C may be used for pre-selection of boars producing sperm with an enhanced tolerance to cold shock.

Effects of handling and storage on potassium concentration in plasma and serum samples obtained from cats.

OBJECTIVE: To compare potassium concentrations in feline plasma and serum samples analyzed promptly after collection or after 20 to 28 hours of refrigerated storage. ANIMALS: 41 cats. PROCEDURES: A venous blood sample was obtained from each cat. Aliquots were placed in 2 tubes without anticoagulant (blood was allowed to clot to derive serum) and 2 tubes with heparin (to derive plasma). One serum and 1 plasma sample were kept at room temperature and analyzed within 60 minutes after collection (baseline); the other serum and plasma samples were analyzed after 20 to 28 hours of refrigerated storage. At both time points, serum and plasma potassium concentrations were measured. RESULTS: Median baseline serum potassium concentration (4.3 mmol/L) was significantly higher than median baseline plasma potassium concentration (4.1 mmol/L). The median difference between those values was 0.4 mmol/L (95% CI, 0.2 to 0.5 mmol/L). Compared with their respective baseline measurements, the median serum plasma concentration (4.8 mmol/L) and median plasma potassium concentration (4.6 mmol/L) were higher after 20 to 28 hours of refrigeration. CLINICAL RELEVANCE: Results indicated that with regard to potassium concentration in feline blood samples, clotting or refrigerated storage for 20 to 28 hours results in a significant artifactual increase. Detection of an unexpectedly high potassium concentration in a cat may represent pseudohyperkalemia, especially if the blood sample was placed in a no-additive tube, was stored for 20 to 28 hours prior to analysis, or both.

Osmotic tolerance of rabbit spermatozoa is affected by extender composition and temperature.

Sperm osmotic adaptability to anisosmotic conditions is important for sperm epididymal maturation, motility activation at ejaculation, and female tract colonization, or for conducting technological procedures such as cryopreservation. Several factors affect this adaptability, including the fluid composition that contributes to water flow dynamics, and the temperature at which osmotic stress is initiated. This study was designed to investigate the effect of medium composition (electrolyte- or sugar-based extender) and temperature (25 and 5 °C) on rabbit sperm adaptability to anisosmotic conditions. Rabbit spermatozoa, therefore, were diluted at both temperatures (25 and 5 °C) in electrolyte- or sugar-based media at increasing osmotic conditions (100 to 1,000 mOsm/kg), and values for sperm variables (sperm kinetics, membrane integrity, mitochondrial membrane potential) were estimated as endpoints. Sperm kinetics seemed to be more sensitive to osmotic stress than membrane integrity or mitochondrial function. The effect of moderate hypoosmotic stress did not differ when there was use of sugar- and electrolyte-based extenders at 25 °C (P > 0.05). In hyper-tonic conditions at 25 °C, the sugar-based extender was more effective in protecting sperm membrane integrity and mitochondrial function (P < 0.05). The lesser temperature made the differences more relevant because of the detrimental effect of hyperosmotic stress was more evident in the electrolyte-based extender at 5 °C (P < 0.05). The results from this study indicated rabbit spermatozoa have different adaptability to anisosmotic conditions induced by sugar- and electrolyte-based media and that the temperature at which the osmotic stress is initiated affects the cellular response.

Investigation of the effects of storage with preservatives at room temperature or refrigeration without preservatives on urinalysis results for samples from healthy dogs.

OBJECTIVE: To compare urinalysis results for canine urine samples stored in preservative-containing tubes at room temperature (20°C to 25°C [68°F to 77°F]) or refrigerated at 4°C (39.2°F) in plain glass tubes with results for the same samples immediately after collection. SAMPLES: Urine samples from 20 healthy dogs. PROCEDURES: Urine samples (1/dog) were divided into 6 aliquots (3 in preservative-containing tubes and 3 in plain glass tubes). Preservative-containing tubes were stored at room temperature and plain glass tubes were refrigerated. Urinalysis was performed 0, 24, and 72 hours after collection. Results for both storage conditions were compared with results for a reference sample (the 0-hour [immediate post-collection] aliquot in a plain glass tube) by Spearman correlation analysis with pairwise tests for selected variables. RESULTS: Physical variables (urine color and turbidity with and without centrifugation) for both storage conditions had high (rs = 0.7 to 0.9) or very high (rs = 0.9 to 1.0) degrees of positive correlation with reference sample results at all time points, except for color at 24 hours. Similar results were found for all biochemical variables with storage up to 72 hours. For microscopic characteristics, correlation with reference sample results ranged from low or nonsignificant to very high under both storage conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that if a delay in urinalysis is expected, use of the preservative-containing tubes evaluated in this study may be a viable option for sample storage. Further research is warranted to assess direct comparability of results to those of freshly collected samples and use of these tubes to store samples from dogs with conditions affecting the urinary tract.

An alternative cold chain for storing and transporting East Coast fever vaccine.

East Coast fever (ECF) is an often fatal, economically important cattle disease that predominantly affects eastern, central, and southern Africa. ECF is controlled through vaccination by means of simultaneous injection of oxytetracycline and cryogenically preserved stabilate containing live, disease-causing parasites. Storage and transportation of the stabilate requires liquid nitrogen, a commodity that is commonly unreliable in low-resource settings. Here we show that storage of conventionally prepared stabilate at -80 °C for up to 30 days does not significantly affect its ability to infect cultured peripheral blood mononucleated cells or live cattle, suggesting an alternative cold chain that maintains these temperatures could be used to effectively manage ECF.

Conservation of the internal quality of eggs using a biodegradable coating.

This study aimed to evaluate the effect of a pectin biofilm on the preservation of refrigerated and unrefrigerated eggs during 5 wk of storage based on egg weight loss, albumen height, Haugh unit (HU), and the yolk index (YI). A total of 1,200 nonfertile eggs from GLK Bankiva laying hens (40 wk of age), which were freshly laid and came from a single collection, were obtained from a model poultry rearing system (Planaltina, Federal District, Brazil) that meets all animal welfare criteria. The experimental outline was entirely randomized, with 20 treatments in a factorial scheme of 2 × 2 × 5, with 2 biofilm treatments (with and without) × 2 storage temperatures (refrigeration: 5°C and ambient: 25°C) × 5 storage periods (7, 14, 21, 28, and 35 d), with 12 repetitions per treatment. Starting from the third storage week, increased weight loss (%) was observed in noncoated eggs (4.46 ± 1.06; 5.61 ± 1.37; 6.93 ± 1.66%) compared with biofilm-coated eggs (3.57 ± 1.26; 4.74 ± 1.8; 6.05 ± 2.21%), respectively. The HU variation in the pectin-coated eggs (86.84-78.02) was smaller than that in the noncoated eggs (83.01-64.36) between the beginning (7 d) and the end (35 d) of the experimental period. Eggs with and without biofilm stored in the refrigerator presented average HU values of 91.26 ± 6.27 and 88.35 ± 6.96, respectively. In contrast, when kept at room temperature, eggs with the coating presented higher HU values (71.27 ± 10.78) than eggs without the coating (59.11 ± 15.97). Coated eggs (0.37 ± 0.16) showed higher YI values than noncoated eggs (0.35 ± 0.16). A pectin-based biofilm effectively maintained egg quality during the 35 d of storage.

Effects of gametes post-activation, egg storage and sperm-egg ratio on in vitro fertilization outcomes in the brown-marbled grouper (Epinephelus fuscoguttatus).

Due to some biological characteristics of the brown-marbled grouper, there are challenges in aquaculture enterprises focused on production of this fish. With the aim of facilitating captive breeding in brown-marbled grouper, effects on gamete post-activation, post-stripping egg storage and sperm to egg ratio on fertilization success were investigated. Results indicated eggs were fertile for 4 min after placement in seawater, thereafter, there was a reduction in fertilization. Initial curvilinear velocity (VCL) and total motility of fresh sperm was 129.3 ±â€¯3.7 µm/s and 92.9 ±â€¯2.4 %, both of which decreased as time elapsed post-activation, however, there was no significant difference in fertilization until 6 min post-activation of sperm. Compared with fertilization and hatching rates in eggs used immediately after collection (95.0 ±â€¯2.8 % and 90.1 ±â€¯4.2 %), in vitro storage of eggs had no detectable effect on fertilized egg and hatching rates until 60 min post-stripping (92.0 ±â€¯2.8 % and 89.9 ±â€¯2.7 %), indicating that immediate use of brown-marbled grouper eggs for fertilization is not necessary in aquaculture hatcheries. With regard to optimizing the sperm to egg ratio, there was acceptable fertilization with ratios ranging from 103 to 107, whereas there was a marked decrease in fertilization rate when there were lesser sperm concentrations. When the combined results from this study are considered, there could be improvements of artificial fertilization efficiency in the brown-marbled grouper with utilization of these findings in aquaculture enterprises focused on production of this fish.