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  • The birds used in this study were of 12 Spanish chicken breeds (Black-Barred Andaluza, Black-Red Andaluza, Blue Andaluza, Black Castellana, Buff Prat, White Prat, Red-Barred Vasca, Red Villafranquina, Birchen Leonesa, White-Faced Spanish, Quail Castellana and Quail Silver Castellana). One hundred forty four roosters (12 of each breed), all of which were one year old at the beginning of the experiment were used for the collection of semen. In addition, hens of White Prat and Black-Red Andaluza breeds (30 hens per breed) were later used for insemination experiments. All animals were housed under natural photoperiod and temperature conditions in two 12 m2 sand-floor pens with partial roof cover at the El Encín Research Station (Madrid, Spain, 40° 31’ N). These birds were raised as part of the INIA’s genetic resources conservation program [28, 29]. All birds were fed a commercial feed containing 16% CP, 2700 kcal of ME/kg, 3.5% Ca and 0.5% available P over the entire experimental period. Animals were handled according to procedures approved by the INIA Ethics Committee (Órgano Regulador de los Comités de Ética de Experimentación Animal, reference number ORCEEA 2016/001) and were performed in accordance with the Spanish Policy for Animal Protection (RD53/2013), which conforms to European Union Directive 86/609 regarding the protection of animals used in scientific experiments. The amino acid profile and total proteins in seminal plasma were studied within each breed. A pool of seminal plasma for each breed was obtained every month, from August to November, and amino acid and total protein analysed in them (n = 48; 4 per breed). Mean sperm concentrations were also evaluated within each breed. To investigate the role of seminal plasma on cryoresistance of rooster sperm, diluted pooled semen samples (n = 168; 14 replicates per breed x 12 breeds) collected from June to November, were divided into two aliquots. One aliquot was frozen with presence of seminal plasma, and the other one was frozen after removal of seminal plasma by centrifugation. Sperm variables were analysed before and after freezing-thawing. The fertilization capacity of frozen semen from one ‘good freezer’ and one ‘bad freezer’ breed, as judged from the obtained in vitro post-thaw sperm assessments, was estimated in an artificial insemination (AI) experiment from the percentage fertile eggs resulting from two consecutive intravaginal AI, three days apart, of a total of 60 hens (30 per breed). Hens of each breed were used for testing fertilizing ability of frozen-thawed semen of the two mentioned breeds; i.e. 15 hens belonging to good freezer breed were inseminated with semen of good freezer, and the remaining 15 with semen of bad freezer; the same criterion was used for the hens of bad freezer breed. Semen collection, management and freezing: Semen was collected twice weekly over the study period, in 15-mL graduated centrifuge tubes (Sterilin) using the massage technique described by Burrows and Quinn [30]. Pools of semen for each breed were made on each occasion. Samples were managed differently, depending on whether they were used for seminal plasma amino acid analysis, or for freezing. Seminal plasma for amino acid assay was obtained by centrifugation of pooled raw semen at 1400g for 30 min. The plasma was evaluated by microscopy to ensure the absence of cells. If any cell were seen, a second centrifugation was made. The pellet was discarded. When semen samples were used in freezing experiments, each pool of semen was immediately diluted 1:1 (v/v) at field temperature using a Lake-Ravie medium [31] composed of sodium glutamate (1.92 g), glucose (0.8 g), magnesium acetate 4H2O (0.08 g), potassium acetate (0.5 g), polyvinylpyrrolidone (Mr 10 000; 0.3 g) and 100 mL H2O (final pH 7.08, final osmolality 343 mOsm/kg; hereinafter referred to as Lake and Ravie medium). This diluted, pooled semen was then immediately placed at 5°C, transported to the laboratory, and sperm concentration and sperm variables (sperm motility variables, plasma membrane integrity) examined (within 45 min of collection). Afterward, each pool was divided into two aliquots. One aliquot, diluted as required with Lake and Ravie medium to a concentration of 1200 × 106 sperm/mL (aliquot with presence of seminal plasma). In the other one (aliquot without seminal plasma) the seminal plasma was removed by dilution with Lake-Centri diluent (1:4 v/v) and centrifugation at 600 g during 20 min prior to freezing. Briefly, the Lake-Centri medium was composed of 1000 ml H2O, 1.28 g potassium citrate tribasic monohydrate, 19.2 g sodium-L-glutamate, 6.0 g D-fructose, 5.0 g TES, 5.1 g sodium acetate trihydrate, 0.8 g magnesium acetate tetrahydrate, and 5.2 ml of 1N sodium hydroxide (340–350 mOsm/kg, pH = 7.0–7.2). The pellet obtained was reconstituted with Lake-Ravie medium. Both aliquot with and without seminal plasma were diluted with Lake-Ravie medium to a final concentration of 1200 × 106 sperm/mL. Pure (≥99%) glycerol (GLY) was then added to the diluted samples, to leave a final 8% concentration (vol/vol), and equilibrated for 10 min at 5°C. After equilibration, the samples were loaded into 0.25 mL French straws and then frozen in two steps, i.e., from 5°C to −35°C at 7°C/min, and then from −35°C to −140°C at 60°C/min [32]. Freezing was performed using a Computer Freezer-Icetube 1810 freezer unit (Minitüb, Tiefenbach, Germany). The frozen straws were then plunged into and maintained in liquid nitrogen (at -196°C) until thawing. For thawing, the straws were warmed for 3 min in a water bath at 5°C. Amino acid and total protein assay: Seminal plasma obtained for amino acid assay (see above) was immediately stored at -20°C until determination of the seminal plasma free amino acid composition. Separation and determination of amino acid was made by ion exchange column chromatography [33,34]. Briefly, the samples were initially precipitated with three volumes of ice-cold acetone and incubated for 2h at—20°C. After centrifugation, the supernatant was removed and freeze dried in a speed vac. The pellet was redissolved in citrate buffer and applied to an ion exchange chromatography amino acid analyzer (Biochrom 30) using post column derivatization with ninhydrin. The ninhydrin reacts with amino acids forming a dye complex. Total protein was assessed by the Coomassie (Bradford) Protein Assay Kit (Thermo Scientific). The seminal plasma was diluted 10 times with Milli-Q water, then, 0.03 mL of the diluted plasma was mixed with 1.5 mL of the Coomassie reagent. The samples were incubated 10 min at room temperature and were analysed by measuring the absorbance at 595 nm (Agilent 8453 Spectrophotometer). The protein concentration was determined by a BSA standard curve with a linear working range of 25–500 μg/mL. Sperm concentration and motility were assayed using a computer-aided sperm analyses (CASA) system coupled to a phase contrast microscope (Nikon Eclipse model 50i; Nikon Instruments Europe B.V., Izasa S.A.; negative contrast) and employing Sperm Class Analyzer (SCA, Barcelona, Spain) v.4.0. software (Microptic S.L., Barcelona, Spain) [35]. For motility analysis, sperm samples were diluted to a concentration of approximately 40 million sperm/ml and loaded onto warmed (38°C) 20 μm Leja 8-chamber slides (Leja Products B.V., Nieuw-Vennep, The Netherlands). The percentage of motile spermatozoa and the percentage showing progressive motility were recorded. Sperm movement characteristics—curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF)—were also recorded. Three progression ratios, expressed as percentages, were calculated from the velocity measurements described above: linearity (LIN = VSL/VCL x 100), straightness (STR = VSL/VAP x 100), and wobble (WOB = VAP/VCL x 100). A minimum of three fields and 500 sperm tracks were evaluated at a magnification of 100x for each sample (image acquisition rate 25 frames/s). Propidium iodide (PI) and SYBR-14 were used as fluorochromes in the examination of membrane integrity [36]; 200 cells were examined using an epifluorescence microscope at 400× (wavelength: 450–490 nm). All sperm variables were measured again for each pool after their eventual thawing. In addition, DNA integrity was also assessed in fresh sperm and after freezing-thawing by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). For this, the kit “In Situ Cell Death Detection” (Roche, Basel, Switzerland) was used following manufacturer’s instructions with minor changes in order to adapt the technique to the analyses of rooster sperm. Briefly, each sperm sample was diluted to 12 x 106 spermatozoa/mL in 4% paraformaldehyde. Subsequently 10 μL of this dilution were placed on a glass slide and left to dry. Then, the spermatozoa were permeabilized with 0.1% of Triton X-100 in PBS. After a wash in PBS, fragmented DNA was nick end-labelled with tetramethylrhodamine-conjugated dUTP by adding 10 μL of the working solution provided by the kit–containing the substrates and the enzyme terminal transferase–on the sample. The reaction was conducted incubating the slides in a humid box for 1 h at 37°C. After a wash with PBS the nucleus were counterstained with Hoechst at 0.1mg/mL in PBS for 5 min in the dark. Following an additional wash with PBS the slides were mounted using Fluoromount (Sigma-Aldrich, MO, USA) and observed under fluorescent microscopy (Eclipse E200, Nikon, Japan). Percentages of positive TUNEL spermatozoa (TUNEL+) per sample were recorded by counting a minimum of 200 spermatozoa per microscopy preparation, using an epifluorescence microscope at 400× (wavelength: 510–560 nm). Cryoprotectant removal and artificial insemination: White Prat and Black-Red Andaluza breeds were chosen for the insemination trial as examples of ‘good freezers’ and ‘bad freezers’, respectively on the basis of the post-thaw in vitro sperm assessments. Glycerol was removed prior to AI. Straws of semen frozen were thawed, and the thawed semen was progressively diluted with four volume parts of Lake Centri medium at 5°C by successively adding 0.07, 0.18, 0.33, 0.6, 1.24, and 1.58 volumes of medium to one volume of semen (2 min intervals). These samples were then centrifuged at 600xg for 10 min, the supernatant solution discarded, and the pellet resuspended (to the original volume of the thawed semen) in Lake and Ravie medium (method adapted from Mocé et al. [37]). All inseminations (see above) were performed between 12:00 h and 14:00 h. AI procedures involved 300 million sperm /female at each insemination. Eggs were collected from day two after the first AI until 3 days after the second AI. Fertility (% fertile/incubated eggs) was determined by candling the eggs (n = 144) on day 7 of incubation. Clustering by the amino acid content in seminal plasma of each breed was performed using the iterative k-technique to classify the amino acids into three clusters. Statistica software (TIBCO Software Inc. Palo Alto, CA, USA) specifically uses Lloyd's method to implement the k-Means algorithm [38]. The right number of clusters was determined by a v-fold cross-validation algorithm included in the Statistica package. Briefly, this method divides the overall sample into a number of v folds (v value: The default value is 10, the minimum is 2, and the maximum is 999). The same type of analysis is then successively applied to the observations belonging to the v-1 folds (training sample), and the results of the analyses are applied to sample v (testing sample) to compute an index of "predictive validity". Variables with a skewed distribution were arcsine-transformed (sperm variables), log-transformed (proteins) or submitted to box-cox transformation (amino acids) before statistical analysis. The influence of breed on amino acid and total protein were analysed by one way ANOVA, following the statistical model xij = m + Ai + eij, where xij = the measured variable (amino acid or total protein), m = the overall mean of x, Ai = the effect of breed (i = 1–12), and eij = the residual (j = 1–4). A Tukey post hoc analysis was performed to compare the differences between means of amino acids. Correlations between amino acids and sperm TUNEL+ and between amino acids and sperm viability were determined by the Spearman test; data of all breeds were included in the correlation analysis. The influence of breed and seminal plasma on frozen-thawed sperm variables were analysed by ANOVA, following the statistical model xijk = m + Ai + Bj + ABij + eijk, where xijk = the measured sperm variable, m = the overall mean of variable x, Ai = the effect of breed (i = 1–12), Bj = the effect of seminal plasma (j = 1–2), ABij = the interaction between A and B, and eijk = the residual (k = 1–14). A post hoc Newman-Keuls analysis was performed to compare the differences in mean sperm variable values between breeds and treatments (with and w/o seminal plasma). Comparisons between fresh and frozen-thawed sperm variables were made using a paired t-test. Identification of good and bad freezer was made by clustering (k-means cluster analysis; see above) the differences between percentage of sperm viability before and after freezing of each breed. The association among fertility rate and semen from good and bad freezers was assessed using the Chi-squared test. Data were expressed as means ± S.E. All statistical calculations were made using TIBCO Statistica software v.13.3 (TIBCO Software Inc.).
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