Beneficial Effects of Rhodotorula sp. C11 on Growth and Disease Resistance of Juvenile Japanese Spiky Sea Cucumber Apostichopus japonicus

The purpose of this study was to evaluate the effects of dietary administration of the live yeast, Rhodotorula sp. C11, on growth and disease resistance against Vibrio splendidus infection in juvenile Japanese spiky sea cucumber Apostichopus japonicus. Sea cucumbers were fed diets containing Rhodotorula sp. C11 at 0 (control), 10⁴, 10⁵, and 10⁶ CFU/g of feed for 45 d. There were three replicate tanks per dietary treatment. The specific growth rates were higher in all sea cucumbers treated with Rhodotorula sp. C11 than in the controls. Following a challenge with V. splendidus NB13, the cumulative prevalence and mortality of sea cucumbers fed diets supplemented with Rhodotorula sp. C11 were lower than in animals fed the basal diet. In sea cucumbers fed diets supplemented with Rhodotorula sp. C11 for 42 d, the only viable yeast found in the intestine was Rhodotorula sp. C11, which had counts of 1.58–1.98 × 10⁴ CFU/g. No yeast was isolated from the intestine of animals fed the basal diet. For the colonization study, 20 sea cucumbers from each dietary treatment were removed to separate tanks and fed the control diet from day 16 to day 46. The viable yeast (Rhodotorula sp. C11) counts in the intestine decreased to 60–80 CFU/g by day 37. Moreover, as demonstrated by denaturing gradient gel electrophoresis, Rhodotorula sp. C11 colonization of the intestine could be detected until day 46. The differences in culture and PCR-denaturing gradient gel electrophoresis may be due to differences in the sensitivity of both methods. The present result showed that Rhodotorula sp. C11 was able to successfully colonize the intestine of juvenile Japanese spiky sea cucumbers by dietary supplementation, which improved its growth and disease resistance.

Received August 11, 2014; accepted November 14, 2014     

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Received February 24, 2015; accepted September 7, 2015     

Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene

Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus–specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate–labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP–LFD technique for V. alginolyticus was 1.8 × 10² CFU/mL while that of PCR was 1.8 × 10³ CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10³ CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP–LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP–LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.

Received November 11, 2014; accepted March 29, 2015     

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP–calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10² F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP–calcein method had 97% homology with the DNA sequence of F. columnare.

Received May 21, 2014; accepted August 10, 2014     

Growth Inhibition of Bacterial Fish Pathogens and Quorum-Sensing Blocking by Bacteria Recovered from Chilean Salmonid Farms

The main goal of this study was to find bacterial isolates with the ability to inhibit the growth of the fish pathogens Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium psychrophilum and to inhibit the blockage of the quorum-sensing (QS) system. A total of 80 gram-negative strains isolated from various freshwater Chilean salmonid farms were studied. We determined that 10 strains belonging to the genus Pseudomonas inhibited at least one of the assayed fish pathogens. Of these, nine strains were able to produce siderophores and two strains were able to inhibit the growth of all assayed pathogenic species. When the 80 strains were examined for QS-blocking activity, only the strains Pseudomonas sp. FF16 and Raoultella planticola R5B1 were identified as QS blockers. When the QS-blocker strains were analyzed for their ability to produce homoserine lactone (HSL) molecules, thin-layer chromatography analysis showed that both strains were able to produce C6-HSL– and C8-HSL–type molecules. Strain R5B1 did not show growth inhibition properties, but strain FF16 also led to inhibition of growth in A. hydrophila and F. psychrophilum as well as to siderophore production. Pseudomonas sp. FF16 exhibited potentially useful antagonistic properties and could be a probiotic candidate for the salmon farming industry.

Received July 31, 2014; accepted December 17, 2014     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

Molecular weight and amino acid composition of fowl delta‐globulin

1. The molecular weight of delta‐globulin was estimated chromato‐graphically to be about 10,700 daltons.

2. Ultracentrifuge experiments at 2.7 × 10⁵ g gave values of s _{20, W} = (1.49 + 0.16C) × 10^?13 s for the sedimentation coefficient and D_{20 w} = (1.12 + 0.19c) × 10^?6 cm² s^?1 for the diffusion coefficient, c being the protein concentration (g/100 ml).

3. According to the Svedberg equation, these imply a molecular weight of 12,470 daltons, assuming [v_bar] = 0.74 ml/g.

4. Using refractometer measurements of protein concentration it was found that E ^1% _1cm (278 nm) = 5.57 at pH 7.8.

5. From the ultraviolet absorption spectrum of the protein in 0.1 N NaOH it was concluded that the molecule probably contains four tyrosine residues and no tryptophan.

6. On this basis a revised amino acid composition is given.     

Stress and Humoral Innate Immune Response of Gilthead Seabream Sparus aurata Cultured in Sea Cages

Innate and acquired immune responses of Gilthead Seabream Sparus aurata was studied under normal culture and short-term stressful conditions for 18 months in offshore sea cages in Alghero Bay, Italy. Every 45 d, 50 fish were sampled and divided into two groups: fish in the first group (normal culture conditions) were bled after harvesting; fish in the second group were put into a tank under stressful conditions (crowding and confinement) and bled after 2 h. Innate humoral immunity, such as complement-like, hemagglutination, and lysozyme activities, was determined in the sera of both groups. Pathogen challenge was not performed, but the specific humoral immune response was assessed against the most common pathogens affecting cultured fish in Sardinia. Stressed fish, compared with the control, showed a lower lysozyme activity against Vibrio (Listonella) anguillarum, which was not clearly correlated with temperatures. Complement-like activity differed between the first and second half of the study and, at the end of the trial, a slightly higher activity was recorded in the controls than in the stressed fish. Hemagglutination activity was mainly higher in the stressed fish than in control fish. Confinement, crowding, and cold water temperature caused decreased lysozyme activity in short-term stressed Gilthead Seabream compared with those reared normally. The specific humoral immune response, against V. anguillarum, Tenacibaculum mesophilum, Enterococcus Seriolicida, and Aeromonas sobria, fluctuated during the rearing period, particularly during the first year of culture.

Received October 12, 2015; accepted March 24, 2016 Published online August 2, 2016     

Natural resistance of Sri Lankan village chicken to Salmonella gallinarum infection

1. An experiment was conducted to compare the natural resistance of an indigenous breed of local village chickens to Salmonella gallinarum with two commercial breeds: ISA Brown and ISA White layers under experimental conditions.

2. A total of 72 chickens from each of these breeds were randomly distributed to 4 pens to provide equal numbers of two replicate pens maintained as infected and control (uninfected). All chickens in infected groups were inoculated orally with 1 × 10⁸ CFU (1 ml dose) of a field isolate of S. gallinarum, at the age of 8 and 16 weeks given over 5 consecutive days. Growth performance, clinical signs, gross pathological lesions and antibody responses were measured.

3. A significantly higher mortality was observed in the brown layers compared with the white layers, and clinical signs and mortality were absent in village chickens. However, a large number of birds with gross lesions and high antibody titres were detected in village chickens, indicating that birds had the disease subclinically. Commercial breeds had a significantly higher body weight, feed intake and feed conversion efficiency.

4. There was a significantly lower proportion of positive reactors in village chickens in the whole-blood agglutination test (35%) compared to brown (100%) and white (90%) layers even after the second inoculation. Uninfected birds were negative in all groups. The indirect enzyme-linked immunosorbent assay confirmed these observations.

5. These results suggest that the indigenous breed had superior natural resistance to S. gallinarum than the commercial breeds.     

Absorption and deposition of xanthophylls in broilers challenged with three dosages of Eimeria acervulina oocysts

1. An experiment was designed to evaluate the effect of different doses of oocysts of Eimeria acervulina on intestinal absorption and skin deposition of xanthophylls (XAs) in broilers.

2. A total of 192 broiler chickens were randomly assigned to 4 groups: an uninfected control group and three groups inoculated with either 1 × 10², 1 × 10⁴ or 1 × 10⁵ sporulated oocysts of E. acervulina by gavaging at 21 d. There were 4 replicate pens (2 male and 2 female) per group.

3. Plasma xanthophyll (PX) and skin yellowness (SY) were measured in live birds weekly. At 42 d of age, SY was measured in the breast and abdomen after chilling and in the breast 24 h post-processing on refrigerated carcasses.

4. In general, in all challenged treatments, and for the duration of the study, the average PX decreased by 0.02 μg/ml (R² = 61.6%) for every 1000 inoculated oocysts, whereas PX increased by 1.26 μg/ml/d in uninfected birds.

5. The average SY in live birds from 21 to 42 d of age decreased by 0.019 b*/every 1000 oocysts administered, while SY of uninfected controls increased by 0.57 b*/d. It was also noted that in all treatments females had a greater SY (6.17 b*) than males for the duration of the study. The SY of the breast and abdomen was correlated (r = 0.76) in chilled carcasses. Breast SY in 24 h refrigerated carcasses was greater in the control group and for female birds.

6. Oocyst excretion was different between inoculated treatments only on 7 d post-inoculation (PI). Coccidia lesion scores in the duodenum averaged 1+ in infected birds and 2+ in birds given the highest oocyst dose.     

Respiratory phagocytes are implicated in enhanced colibacillosis in chickens co-infected with influenza virus H9N2 and Escherichia coli

1. The aim of this study was to determine the most likely time interval after infection with influenza virus H9N2 for co-infection with Escherichia coli to cause colibacillosis, the importance of lung load of E. coli and the involvement of respiratory phagocytes.

2. Specific pathogen free chickens were inoculated intranasally with 10⁶EID₅₀ of influenza virus or uninfected. After specified time intervals, 10⁷ CFU E. coli or phosphate-buffered saline was inoculated. The presence of lesions, the number of respiratory phagocytes in the respiratory lavage fluid and the E. coli load in the lung were determined after different time intervals.

3. Compared with the number of lesions in chickens receiving only E. coli inoculation, the number lesions in co-infected chickens were increased at 0- and 3-d time intervals, but reduced in the groups at 6- and 9-d intervals between co-infection.

4. At 1–3 d after E. coli inoculation, the number of lesions chickens was correlated with the number of respiratory phagocytes harvested and related to the E. coli load in the lungs at 5 d.

5. These results suggest that the lesions caused by E. coli in chickens were increased within a 0–3 d interval following H9N2 virus inoculation and that this effect is related to the number of respiratory phagocytes.     

Presence and incidence of food-borne pathogens in Australian chicken litter

1.?Litter samples were collected at the end of the production cycle from spread litter in a single shed from each of 28 farms distributed across the three Eastern seaboard States of Australia.

2.?The geometric mean for Salmonella was 44 Most Probable Number (MPN)/g for the 20 positive samples. Five samples were between 100 and 1000 MPN/g and one at 10⁵ MPN/g, indicating a range of factors are contributing to these varying loads of this organism in litter.

3.?The geometric mean for Campylobacter was 30 MPN/g for the 10 positive samples, with 7 of these samples being <100 MPN/g. The low prevalence and incidence of Campylobacter were possibly due to the rapid die-off of this organism.

4.?E. coli values were markedly higher than the two key pathogens (geometric mean 2?0 × 10⁵ colony forming units (cfu)/g) with overall values being more or less within the same range across all samples in the trial, suggesting a uniform contribution pattern of these organisms in litter.

5.?Listeria monocytogenes was absent in all samples and this organism appears not to be an issue in litter.

6.?The dominant (70% of the isolates) Salmonella serovar was S. Sofia (a common serovar isolated from chickens in Australia) and was isolated across all regions. Other major serovars were S. Virchow and S. Chester (at 10%) and S. Bovismorbificans and S. Infantis (at 8%) with these serovars demonstrating a spatial distribution across the major regions tested.

7.?There is potential to re-use litter in the environment depending on end use and the support of relevant application practices and guidelines.     

Thermoneutral zone and resting metabolic rate of broilers

1. Metabolic rate was determined once a week in broilers from a commercial source, from 1 to 63 d of age.

2. The equations relating minimal resting metabolic rate (oxygen consumption, ml/bird h,y) and body weight (W) were: males 45 to 497 g, y = 3.2 W^0.882 597 to 3000 g, y = 40.5 W^0.483; females 45 to 514 g, y = 2.52 W^0.881; 514 to 2500 g,y = 12.3 W^0.627.

3. The relationship between lower critical temperature (T_cl , °C,y) and age (d, x) may be described by the following equations: chicks 1 to 21 d, y = 34.2 ‐ 0.32 x; 14 to 63 d,y = 49.4 × ^?0194.

4. The relationship between T_cl and W may be described by one equation for both sexes between 100 and 3000 g, y = 62.15 W^?0.135..

5. The equations for T_cl and data for upper critical temperature (T_cu ) could be used to obtain maximal performance from broilers, with reduced costs, by providing a suitable environment related to age or body weight.     

Bacterial Analysis of Fertilized Eggs of Atlantic Salmon from the Penobscot,Naraguagus, and Machias Rivers,Maine

Serious losses have occurred at the U.S. Fish and Wildlife Service, Craig Brook National Fish Hatchery, East Orland, Maine, among eggs that were taken from Atlantic Salmon Salmo salar, which were held as captive broodfish during their returns to the Penobscot River, Naraguagus River, and Machias River to spawn. Bacterial isolations were attempted from external surfaces and the internal contents of individual eggs. Externally and in all cases, Pseudomonas fluorescens was the predominant bacterium associated with the surface of all eggs. These bacteria were resistant to a surface treatment of 1,667 ppm formalin for 15 min and, therefore, the monoclonal nature of P. fluorescens on egg surfaces was considered to result from its ability to resist the germicidal activity of formalin administered for antifungal treatments. Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was isolated from the interior of 23.6, 18.1, and 29.2% of the dead Atlantic Salmon eggs from Penobscot River egg lots A-98, A-100, and A-101, respectively, and concentrations of this pathogen ranged from 1.0 × 10³ to >5 × 10⁸ CFU per gram of dead egg. Flavobacterium psychrophilum was also isolated from 8.3, 26.7, and 10.0% of the dead eggs from Naraguagus River egg lots N-158, N-161, and N-163, respectively, in which concentrations of this organism ranged from 1.0 × 10³ to 7.5 × 10⁸ CFU per gram of egg. This bacterium was also isolated from within 18.3% and 3.3% of the dead eggs from Machias River egg lots M-128 and M-142, respectively, and its concentrations ranged from 1.0 × 10³ to 1.5 × 10⁸ CFU per gram of egg. The finding of F. psychrophilum from within these eggs is indicative of this pathogen's widespread and persistent prevalence in Atlantic Salmon in New England.

Received December 19, 2014; accepted May 4, 2015     

Growth performance and meat quality of broiler chickens supplemented with Rhodopseudomonas palustris in drinking water

1. The effect of the bacterium, Rhodopseudomonas palustris, on the growth performance and meat quality of broiler chickens was investigated.

2. A total of 900-d-old Arbor Acres broilers were allocated to three experimental treatments for 6 weeks. Chicks were administered with R. palustris in drinking water as follows: (i) control group without R. palustris; (ii) treatment 1 (R1) with R. palustris of 8 × 10⁹ cells per chick per day in drinking water; (iii) treatment 2 (R2) with R. palustris of 1.6 × 10¹⁰ cells per chick per day in drinking water.

3. The results showed that, compared with that of control, both groups of R. palustris treatment increased daily weight gain and improved feed conversion ratio of broiler chickens significantly during the whole growing period of 6 weeks.

4. Both total and glutamic acid contents of chicken breast fillet in R. palustris treatment R2 were higher, while the fat content was lower, than those of the control group. Furthermore, R. palustris treatments also improved sensory attributes of chicken breast fillet.

5. As a probiotic providing rich nutrients and biological active substances, R. palustris administration in drinking water displayed a growth promoting effect and improved meat quality of broiler chickens.     

Microbiological survey of five poultry processing plants in the UK

1. Neck skin samples were taken from chickens and turkeys at all the main stages of processing to monitor changes in total viable count (TVC) and counts of coliforms and pseudomonads.

2. Processing reduced TVC by up to 100‐fold. Geometric mean counts after packaging were log₁₀ 4.4 to 5.3 CFU/g whilst corresponding counts of coliforms were 2.7 to 3.8 CFU/g.

3. Increases in mean TVC or coliforms as a result of either defeathering or evisceration did not exceed 0.6 log.

4. Pseudomonads represented only a minor fraction of the initial microflora of the bird and were often reduced by scalding to a figure which could not be detected by direct plating of samples; however, subsequent contamination resulted in means between log₁₀ 2.9 and 4.0 CFU/g for packaged carcases.

5. Although Staphylococcus aureus was readily isolated from defeathering equipment, mean counts from defeathered carcases were always below log₁₀ 3.0 CFU/g.     

Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria

1. The aim of this work was to compare conjugated linoleic acid (CLA) concentrations in chickens supplemented with 4 American Tissue Culture Collection (ATCC) bacterial strains, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus casei and Lactobacillus fermentum, and 4 isolates of Lactobacillus reuteri from camel, cattle, sheep and goat rumen extracts.

2. Micro-organisms were grown anaerobically in MRS broth, and 10⁶ CFU/ml of bacteria were administered orally to mixed-sex, 1-d-old broiler chickens weekly for 4 weeks and to 23-week-old layer hens weekly for 6 weeks.

3. The 4 strains were evaluated for their effects on synthesis of CLA in hen eggs and broiler meat cuts.

4. Administration of pure Lactobacillus and isolated L. reuteri strains from camel, cattle, goat and sheep led to significantly increased CLA concentrations of 0.2–1.2 mg/g of fat in eggs and 0.3–1.88 mg/g of fat in broiler chicken flesh homogenates of leg, thigh and breast.

5. These data demonstrate that lactic acid bacteria of animal origin (L. reuteri) significantly enhanced CLA synthesis in both eggs and broiler meat cuts.     

Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain.

2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2 × 10⁹ cells/ml in pre-freezing extender; equilibration of diluted semen at 5°C, 20 vs 40 min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5°C, 1 vs 30 min; thawing at 60°C for 10 vs 50°C for 30 sec. Spermatozoa viability (EtBr exclusion procedure – stress test), mobility (Accudenz® swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen.

3. The lower SWC (1.5 × 10⁹ cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively.

4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol.

5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed.

6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa.