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The aim of this study was to compare the effect of two permeant-cryoprotectants, dimethylacetamide (DMA) and N-methylacetamide (NMA) used at different concentrations (0%, 2%, 4%, 6%) on the quality and fertility of post-thaw rooster semen. Ejaculates were processed in 7 treatments: Lake pre-freezing+0.1 M trehalose (LPF-T) (control treatment), LPF-T+2% DMA, LPF-T+4% DMA, LPF-T+6% DMA, LPF-T+2% NMA, LPF-T+4% NMA, LPF-T+6% NMA. Sperm quality [sperm membrane integrity (SMI), motility and kinetic parameters] was assessed before and after cryopreservation. Fertility and embryo viability were recorded. Increasing both DMA and NMA concentration from 2 to 6% improved SMI, total motile sperm, progressive motile sperm (PMS), VCL, VSL and VAP values. PMS recovery rates were significantly the highest in 6% DMA, 4% NMA and 6% NMA treatments. Semen cryopreserved with DMA produced the best fertility and embryo viability at 6%; progressive lower values were recorded at lower concentrations, with no viable embryos at 2%. Semen cryopreserved with NMA showed the best fertility values at 2% and lower values were recorded at higher concentrations; live embryos were found in all NMA treatments. Finally, NMA and DMA showed a similar positive concentration dependent effect of the quality of cryopreserved semen. NMA, not DMA, provided the highest fertility and embryo viability values at the lowest 2%. Therefore, the use of NMA is recommended in order to reduce the cryoprotectant concentration, with a concomitant reduction in the risk of toxicity, providing at the same time the adequate cryoprotective action to obtain viable embryos after artificial insemination of cryopreserved chicken semen., (Copyright © 2022. Published by Elsevier Inc.)

The aim was to compare the effect of dimethylacetamide (DMA) and N-methylacetamide (NMA) concentrations on the quality and fertility of post-thaw chicken semen. Ejaculates were obtained from 30 Hi-Line White roosters and processed according to the following treatments: lake pre-freezing extender + 0.1 M trehalose (LPF-T) + 6% DMA (control treatment), LPF-T + 9% DMA, LPF-T + 6% NMA, and LPF-T + 9% NMA. Sperm quality (viability, motility, and kinetic traits) was assessed before and after cryopreservation. A total of 15 laying hens per treatment were inseminated to assess fertility and embryo viability. Sperm cryopreserved in presence of DMA had significantly better in vitro quality compared to NMA, showing the highest proportion of viable and progressive motile sperm recovered after thawing. Furthermore, proportion of progressive motile sperm and the VCL, LIN, ALH, and WOB mean values were significantly improved in semen samples frozen/thawed with 6% compared to 9% cryoprotectant concentration. However, the best cryoprotective action on sperm quality played by DMA and the lowest cryoprotectant concentration did not translate into a concomitant advantage in in vivo semen fertility that showed no differences between cryoprotectant and cryoprotectant concentration treatments. Finally, the cryoprotectant DMA and NMA showed an opposite effect on embryo viability in comparison with the effect played on in vitro semen quality, being NMA more efficient than DMA on preserving viable embryos. The present results suggest the urgency to further decrease the cryoprotectant concentration in poultry semen freezing procedures and to assess the specific toxic effect of cryoprotectant on sperm integrity, fertility, and embryo development., (© 2019 Poultry Science Association Inc.)

The study aimed to characterize meat quality traits of Milanino chickens reared according to a specific free-range farming program. A total of 120 birds was reared straight-run in outdoor pens (8 m2/bird) from 35 d of life and fed ad libitum a low (16%) protein diet. At 180 d of age, 20 birds (10 birds/sex) were slaughtered, and carcass weight data were recorded. After processing, carcasses were refrigerated at 4°C for 24 hours. Then, the right breast and thigh with skin were collected and color parameters, pH, water-holding capacity (WHC), and chemical composition were determined. The left breast and thigh were stored at -20°C until cooking loss and tenderness evaluation. Milanino was confirmed to be a heavy breed with a sexual dimorphism in relation to adult body weight. A high general carcass yield was recorded. Milanino meat was characterized by high protein and low fat contents compared with the standard broiler meat. Differences in meat composition were recorded according to the sex: females presented higher values of dry matter (breast and thigh), protein (breast), and fat (breast and thigh) contents. The meat with skin presented an intense luminosity, and this trait was higher in the females. The muscle color was characterized by high redness and yellowness indices with differences according to the sex: Higher yellowness index was observed in female carcasses, while higher redness index was detected in male breast samples. The pH muscle values were similar to those reported in other autochthonous breeds. WHC values did not show variation between sexes. In contrast, cooking loss values recorded in thigh samples were lower in males compared to females. The degree of tenderness of Milanino meat was not affected by the sex. However, the potential loss of water and the toughness in Milanino meat were low compared to other local chicken breed meat. The present results support the breeding of Milanino chickens for meat production according to its specific straight-run free-range system.

The aim of this study was to assess the combined effect of dimethylacetamide (DMA) and two non-permeating cryoprotective agents, trehalose and sucrose, on the quality of post-thaw chicken semen. Adult Lohmann roosters (n = 27) were used. Semen was processed according to the following treatments: Lake pre-freezing extender +6% DMA (LPF, control treatment), LPF + 0.1 M trehalose (LPF-T treatment), LPF + 0.1 M sucrose (LPF-S treatment) and LPF + 0.1 M trehalose + 0.1 M sucrose (LPF-TS treatment). Semen was loaded into straws and frozen in nitrogen vapour. Sperm quality (viability, mobility and kinetic parameters) was assessed immediately after thawing (T0) and at 5 (T5), 10 (T10) and 15 min (T15) thereafter. The different cryodiluent combinations significantly affected the kinetic parameters. The presence of trehalose, alone or with sucrose, combined with DMA improved the quality of motion in cryopreserved sperm in comparison to DMA alone (LPF) and DMA with sucrose (LPF-S). In particular, the highest values in linearity (LIN) and wobble (WOB) were measured in the treatment LPF-T. The treatments significantly affected the recovery rate of progressive motile sperm that presented the best value soon after thawing in the LPF-T treatment; moreover, the presence of trehalose, alone (LPF-T) or with sucrose (LPF-TS), significantly improved the recovery rate of progressive motile sperm also at T5 and T10 compared to LPF and LPF-S. The present results show a positive synergic action of DMA and trehalose on motile function of thawed chicken sperm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the study was to standardize the pellet cryopreservation procedure for chicken semen. Mericanel della Brianza male chicken breeders (Italian breed) were used. Pooled semen samples were processed according to the following conditions: (1) dilution in prefreezing extender to 1 versus 1.5 bill cells/mL sperm working concentration (SWC); (2) 6% versus 9% dimethyl acetamide (DMA) concentration (DMAco); (3) 1 versus 30 minutes DMA equilibration (DMAeq) at 4 °C. Sperm viability and motility were assessed in semen (four replicates/treatment) soon after collection (time 0), after DMAeq (time D), and after freezing/thawing (time FT). The recovery rates (%) of viable and motile sperm after freezing/thawing were also calculated. The low SWC (1 bill/mL) and the low DMAco (6%) indicated a positive significant effect on the proportion of motile sperm (1 bill/mL = 53% vs. 1.5 bill/mL = 48%; 6% DMA = 55% vs. 9% DMA = 47%). Very short DMAeq (1 minute) did not significantly change sperm viability during processing (from time 0 to time D) before freezing whatever the DMAco, and, in contrast, the longer DMAeq showed a significant negative effect on sperm viability. The highest proportion of motile sperm was recorded in semen samples diluted to 1 bill/mL and added with 6% DMA; in this condition, DMAeq had no effect (57% 1 minute and 61% 30 minutes). Increasing SWC to 1.5 bill/mL and adding again 6% DMA, a significant effect of DMAeq was observed, and the higher proportion of motile sperm (58% vs. 43%) was recorded after 1 minute DMAeq. A general decrease in sperm motility was shown in semen samples with 9% DMA (47% vs. 55%), and different conditions in SWC and DMAeq were not effective in the prevention of such decrease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at -196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. alpha- and delta-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. delta-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.

The aim of the present experiment was to study the effect of fish oil and Vitamin E rich diets on semen production, sperm functions and composition in broiler breeders. The following parameters were measured: semen volume and concentration, sperm motility and viability, sperm susceptibility to induced peroxidation, sperm lipid and alpha-tocopherol contents. Dietary n-3 PUFA were successfully transferred into spermatozoan phospholipid by fish oil feeding according to the following main features: (a) the C22:6n-3 and C22:5n - 3 contents were increased, but C22:4n-6 remained the peculiar and major polyunsaturate; (b) the content and proportion of total PUFA did not change; (c) the proportional increase of n-3 PUFA was compensated by the decrease of n-6 PUFA, an increase in the proportion of n-9 fatty acids was also found. The sperm content of alpha-tocopherol was doubled increasing the dietary availability of the vitamin to 300 mg/kg of feed. The specific n-3 PUFA and Vitamin E enrichment of chicken sperm affected cell functions. Significant interactions between the two treatments were also found for some parameters. The best sperm quality condition in control sperm (rich mainly in n-6 PUFA) was found supplying 200mg Vitamin E/kg of feed to the male breeders, and in contrast in n-3 rich sperm supplying 300 mg Vitamin E/kg.

The aim of the present study was to characterize the dietary effects of n-3 LC-PUFA and alpha-tocopheryl acetate (vE) on the quality, phospholipid fatty acid composition, alpha-tocopherol content (alpha-T) and in vitro susceptibility to lipid peroxidation in turkey semen. Fertility of fresh semen was also evaluated. Male turkeys were randomly divided and fed either a control diet or a fish oil and vE rich diet (FO diet) from 40 to 60 weeks of age. The FO diet increased the proportion of n-3 fatty acids in spermatozoa and as a consequence the (n-3)/(n-6) ratio also increased. These changes did not affect the proportion of n-9 PUFAs, particularly of C22:3n-9, in semen. The sperm content of alpha-T was dependent by the dietary supplementation of the vitamin and the sperm content was more than doubled supplying 120 mg kg(-1) of feed to the males compared to the 60 mg kg(-1) of feed in the control diet. In agreement with the major content of alpha-T in spermatozoa collected from the FO group were significantly less susceptible to in vitro induced oxidation. The reproductive capacity of the male breeders was not affected by the diet; however the result is considered of some relevance for field conditions where even very small changes have economic interest being applied to large bird population.

The cellular and molecular mechanisms regulating the reuptake of the testicular fluid supporting sperm exiting the testes in the bird are not known. The presence of aquaporins, proteins involved in transmembrane water transport, was investigated. Observations were limited to the ductuli efferentes, collecting ducts, and ductus epididymis. Interestingly all of these ducts were positive for aquaporins-2, -3, and -9 but not aquaporin-7. When positive, aquaporin was observed localized over the whole cell or the apical plasma membrane of the nonciliated cells and the apical plasma membrane and cilia of the ciliated cells. This study is the first to clearly demonstrate the presence of aquaporins-2, -3, and -9 in the epididymal region of any bird. We assume the aquaporins play a role in concentrating the sperm and in the promotion of sperm maturation in the epididymal region.

Oviductal sperm storage tubules (SST), located at the uterovaginal junction, are the primary site of sperm storage in turkeys. Sperm reside within these storage sites and may be released via a dynamic interaction between sperm mobility and a fluid current generated by the SST epithelial cells. In this study, aquaporins 2, 3, and 9 (proteins that form water channels in the plasmalemma of a variety of cells) were immunocytochemically localized within the apical aspect of the epithelial cells that form the SST. These observations support the contention that the SST epithelial cells are capable of water exchange that may interact with sperm residing within the SST.