Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-µg zinc oxide (ZnO), ZnONP50: Lake with 50-µg ZnONP, ZnONP100: Lake with 100-µg ZnONP and ZnONP200: Lake with 200-µg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-µg ZnO and 50- to 100-µg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Effect of freeze-dried quail egg white and yolk addition to semen extender on viability of rooster sperm stored for 6 h at 4°C.

The effect of freeze-dried quail egg white and yolk addition to basic EK extender on morphology and motility of chicken broiler breeder semen was investigated. Fresh pooled semen was divided into eight parts: fresh, undiluted (control), diluted in 1:2 ratio (v/v) with basic EK extender, EK + 200 mg/ml of egg white, EK + 100 mg/ml of egg white, EK + 50 mg/ml of egg white, EK + 100 mg/ml of egg yolk, EK + 50 mg/ml of egg yolk, EK + 25 mg/ml of egg yolk. Semen samples were evaluated 15 min after dilution and after 6 h of storage at 4°C. In the fresh semen, the number of live normal sperm was the highest in semen diluted with EK + 200 mg of egg white and EK + 100 mg of egg yolk, while the highest sperm motility was in the neat semen. Semen storage reduced the number of normal sperm in all analysed semen samples. In the neat semen, the number of normal sperm decreased, in relation to the fresh not-stored samples, by 36.8% (from 72.3% to 35.5%), with EK extender by 9.2%, in samples enriched with egg white, from 8.4% (EK + 200 mg) to 10.0% (EK + 100 mg), and in EK with egg yolk addition, from 1.2% (EK + 50 mg) to 10.6% (EK + 100 mg). The highest percentage of motile sperm was observed in EK extender enriched with 50 mg of egg white (77.1%) and EK + 25 mg of egg yolk (65.3%).

Melatonin Inhibits Testosterone Synthesis in Rooster Leydig Cells by Targeting CXCL14 through miR-7481-3p.

Melatonin has been proved to be involved in testosterone synthesis, but whether melatonin participates in testosterone synthesis by regulating miRNA in Leydig cells is still unclear. The purpose of this study is to clarify the mechanism of melatonin on Leydig cells testosterone synthesis from the perspective of miRNA. Our results showed that melatonin could significantly inhibit testosterone synthesis in rooster Leydig cells. miR-7481-3p and CXCL14 were selected as the target of melatonin based on RNA-seq and miRNA sequencing. The results of dual-luciferase reporter assays showed that miR-7481-3p targeted the 3'-UTR of CXCL14. The overexpression of miR-7481-3p significantly inhibited the expression of CXCL14 and restored the inhibitory role of melatonin testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. Similarly, interference with CXCL14 could reverse the inhibitory effect of melatonin on the level of testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. The RNA-seq results showed that melatonin could activate the PI3K/AKT signal pathway. Interference with CXCL14 significantly inhibited the phosphorylation level of PI3K and AKT, and the inhibited PI3K/AKT signal pathway could reverse the inhibitory effect of CXCL14 on testosterone synthesis and the expression of StAR, CYP11A1 and 3ß-HSD in rooster Leydig cells. Our results indicated that melatonin inhibits testosterone synthesis by targeting miR-7481-3p/CXCL14 and inhibiting the PI3K/AKT pathway.

Effect of dietary n-3 fatty acid and rooibos (Aspalathus linearis) supplementation on semen quality, sperm fatty acids and reproductive performance of aged male broiler breeders.

The purpose of this study was to assess the effects of dietary fish oil (FO) and rooibos supplementation on semen quality, fatty acids composition and reproductive performance of aged male broiler breeders. Seventy-two 47-week-old Ross broiler breeder roosters were randomly assigned to a 2 × 3 factorial arrangements to include two FO concentrations (0% and 2%) and 3 rooibos concentrations (0%, 1.5% and 3%) for 13 weeks consecutive. The different diets affected semen parameters significantly (p < 0.05), except for the semen concentration and abnormality of the sperm. The sperm of the FO and 3% rooibos-treated group showed better motility and viability when compared to the other groups (p < 0.05). The susceptibility of semen to lipid peroxidation was increased in roosters fed the rooibos-free diets (p < 0.05), but it was reduced (p < 0.05) when the diet was supplemented with 1.5% and 3% rooibos. In addition, at 64 weeks, the highest concentration of testosterone was observed in the roosters fed a diet that included 2% FO and 3% rooibos (p < 0.05); however, the difference in testosterone levels between Week 52 and Week 64 was not significant (p > 0.05). The fertility rate of collected eggs from the FO and 3% rooibos group was higher (p < 0.05) than that of the other groups at the end of the experiment. In conclusion, dietary inclusion of FO along with rooibos improved seminal quality and reproduction performance in aged roosters.

Effects of ginger (Zingiber officinale) supplementation on testicular histology, semen characteristic, blood plasma parameters and reproductive performance in aged broiler breeder roosters.

Higher long-chain polyunsaturated fatty acids contents in roosters' sperm plasma membrane along with age-related decrease in antioxidant defense make the spermatozoa very susceptible to lipid peroxidation. Ginger root contains abundant amounts of gingerol, shogaols, gingerdiol and other active compounds, which known as antioxidant compounds to enhance semen quality. The goal of the study was to evaluate the effect of dietary supplementation of ginger root on semen quality, blood chemistry, immune response, testicular histology and reproductive performance of Ross-308 breeder roosters from 47 to 60 weeks of age. The feeding of ginger root resulted in an increase in parameters related to sperm forward motility and seminal total antioxidant capacity (TAC), and following there was a tendency to increase and decrease in seminal superoxide dismutase activity and malondialdehyde concentration, respectively; however, sperm concentration was not affected. There was an increase and tendency to increase in blood total protein and TAC in the supplemented group respectively. The roosters fed ginger supplemented diet had a higher spermiation index; and following there was tendency to increase seminal tubes spermatozoids number (p = 0.056) and repopulation index (p = 0.058). Despite the improved seminal antioxidant status and a tendency to lower embryonic mortality in the ginger-received group, the fertility and hatchability rate of roosters were statistically insignificant. Supplementations of ginger root in ageing rooster's diet had a beneficial effect on sperm motility, seminal antioxidant status and testicular spermiation index.

Effects of dietary supplementation of N-carbamylglutamate on the haematology parameters, secondary sexual characteristics and testicular gene expression in roosters.

A total of 288 11-week-age roosters were used to evaluate the effects of dietary supplementation of N-carbamylglutamate (NCG) on reproductive traits and testicular gene expression. The experimental periods were 12 weeks. All birds were randomly assigned to 4 treatments with 6 replicates per treatment and 12 birds per replicate. Dietary conditions were based on a basal diet and supplemented with 0%, 0.08%, 0.12%, or 0.16% NCG to form C, N1, N2 and N3 groups respectively. Dietary supplementation of NCG had positive effects on the seminiferous tubule parameters, serum gonadotropin-releasing hormone and testosterone levels and the secondary sexual characteristics. Transcriptomics analysis was performed on the testicular tissues between C and N3 groups at the 16-week-age. Genes were mainly enriched in nine pathways, such as cytochrome P450 exogenous metabolism, drug metabolism, steroid hormone synthesis and glutathione metabolism, in which the ZP4 gene, cytochrome P450 family member 11A1 and other genes involved in the maintenance of gonadal function, steroid hormone biosynthesis and metabolism, and so forth, exist differences in expression levels. In summary, dietary supplementation of NCG had positive effects on the reproductive traits of roosters. NCG supplementation improved the development of reproductive traits of roosters by regulating the genes expression in testicular tissues and thus improved the synthesis of reproductive hormones in vivo.

Estimation of the seminal parameters of rooster and its association with fertility traits in synthetic dam line.

The objective of the present study was to assess the seminal parameters of rooster and its association with fertility traits (%), viz., hatchability of the fertile egg set (HFES), hatchability of the total egg set (HTES), and fertility (FERT). The data records pertained to traits of interest were obtained from various registers maintained at Poultry farm, of the Department of Animal Genetics and Breeding, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (India). The relationship between seminal and fertility characteristics was investigated using regression analysis and correlation. Moreover, the efficacy of seminal characteristics to distinguish between roosters with low and high fertility traits was evaluated using linear discriminant analysis (LDA). The findings showed that reproductive traits and seminal characteristics were significantly (P < 0.05) correlated. The LDA showed that the seminal parameters can effectively separate the roosters into those with high and poor reproductive features. It was revealed from LDA that seminal features showed higher classification accuracy for FERT (80.77%). Hatchability is dependent on eggs that have been artificially incubated; hence, these crucial traits are comparatively weaker for HTES (65.38%) and HFES (67.31%). Cross-validation of the seminal parameter LDA corroborated the aforementioned and related conclusions. It is suggested that the studied LDA function may be utilised to choose genotypes with improved reproductive traits based on seminal variables.

Influence of Technological Stages of Preparation of Rooster Semen for Short-Term and Long-Term Storage on Its Quality Characteristics.

There is a problem of declining quality of rooster semen in the "native semen-equilibrium-short-term and long-term storage (cryopreservation)" cycle. The aim of this study was to determine the effects of various methods of preparing rooster semen on its qualitative characteristics, taking into account the method of removing possible contaminants (centrifugation or filtration), and to evaluate the change in the composition of the cytosol of the spermatozoon of the native semen, during equilibration of the diluted semen and during short-term storage. In this study, semen from roosters (n = 22) of the Russian White breed was used. Experiment 1: semen was divided into 3 aliquots: I-was diluted with synthetic cryoprotective medium (1:1 with LCM control, II-was filtered (membrane pore Ø 0.2 µm), and III-was centrifugated (at 3000 rpm for 10 min). Native and frozen/thawed semen was evaluated. Experiment 2: the composition of carbohydrates and polyols of the spermatozoa of native semen was evaluated during equilibration and after storage (3 h). The results of Experiment 1 showed an advantage in the quality of filtered semen compared to centrifuged in terms of progressive motility (41.0% vs. 27.0%) and chromatin integrity (56.6% vs. 33.6%). Results from frozen/thawed samples of filtered semen compared to centrifuged in terms of progressive motility were 25.5% vs. 5.5%, respectively, and in terms of chromatin integrity-83.5% vs. 64.4%, respectively. The results of Experiment 2 showed the main component in the composition of the native spermatozoa cytosol in assessing the content of carbohydrates and polyols was inositol-75.6%. The content of inositol decreased during storage by 6.5 times (from 0.030 mg/mL to 0.007 mg/mL), proposing the role of inositol as the main antioxidant in the cytosol of spermatozoa, which makes it biologically justified to introduce inositol into the composition of synthetic diluents, including cryoprotective ones.

Birchen and Blue Leonesa sperm cryopreservation: a new technique for evaluating the integrity of cockerel sperm membranes.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

Effects of P-nitrophenol exposure on the testicular development and semen quality of roosters.

The widespread use of P-nitrophenol (PNP) as a raw material in pesticides, medicines and dyes has led to environmental pollution. PNP is a well-known endocrine disruptor in mammals and quails. This study investigated the effects of long-term PNP exposure on the testicular development and semen quality of roosters. Pubescent and postpubescent animals were given drinking water supplemented with (0 mg/L, 1 mg/L, 10 mg/L, or 100 mg/L) PNP for eight weeks or sixteen weeks. The relative testis weight, antioxidant index, serum hormone concentration, morphological changes, semen quality and expression of major steroidogenic genes were measured. The results showed that eight weeks of PNP exposure decreased CAT activity and increased H2O2 level in serum and testes in the 10 mg/L and 100 mg/L PNP-treated groups. Detached sperm cells were also found in the testicular tissues of the 100 mg/L PNP-treated group. After sixteen weeks of PNP exposure, daily weight gain, sperm motility, serum testosterone concentration and 3ß1-hydroxysteroid dehydrogenase (HSD3ß1) mRNA expression were decreased in the 100 mg/L PNP-treated group. Some vacuoles in the seminiferous epithelium in the testicular tissues were found in the 10 mg/L and 100 mg/L PNP-treated groups. In conclusion, as an endocrine disruptor, PNP exposure impaired antioxidant capacity, reduced testosterone synthesis, caused morphological changes in testes, and ultimately decreased semen quality in the roosters. The reproductive damage of PNP to roosters depended on the length of exposure time and the administered dose.

Effect of organic selenium dietary supplementation on quality and fertility of cryopreserved chicken sperm.

Oxidative stress due to cryopreservation has been considered as a major factor in sperm damage. Supplementation of the diet with different concentrations of organic selenium has been proposed to improve the quality of fresh and frozen-thawed semen in different breeds of roosters. Sixteen Pradu Hang Dum (Thai native) and 16 Rhode Island Red roosters were used in this study. Four levels of selenium supplementation between 0 and 0.9 ppm were examined. After 14 days of feeding, semen samples were collected twice a week and the fresh semen was evaluated. Then semen from each group was pooled and cryopreserved. The fertility of frozen-thawed semen was determined by inseminating 48 layer hens. Supplementation of diets with 0.3, 0.6 and 0.9 ppm selenium improved the fresh semen in terms of sperm viability and normal morphology (P < 0.01). Sperm concentration increased (quadratically, P < 0.001) with increasing dietary selenium levels. Meanwhile, post-thawed semen quality in terms of sperm motility, viability, live with intact acrosome and functioning mitochondria improved significantly with selenium treatments of 0.6 and 0.9 ppm, and lipid peroxidation was decreased (P < 0.001) and fertility improved (P < 0.05) with those levels of selenium treatment. In addition, there were differences between breeds with respect to some fresh or frozen semen quality parameters (P < 0.05). In conclusion, the breed affected both fresh and frozen semen. Even there were no statistically significant differences in the parameters from groups 0.6 and 0.9 ppm on frozen-thawed semen quality, but the highest sperm concentration was found in 0.6 ppm. Therefore selenium supplementation of diets at 0.6 ppm was recommended to improve the quantity and quality of fresh and frozen semen.

Royal jelly may improve sperm characteristics during preservation of rooster semen: Gene expression of antioxidant enzymes.

Sustainable production and the increasing number of embryonated hatching eggs are critical aspects of the poultry production industry. The present paper aims to appraise the effectiveness of royal jelly (RJ) on the semen characteristics of Native Mazandaran roosters in both liquid and frozen storage conditions. Semen collected from 10 sexually mature roosters and following dilution was supplemented with RJ at 0.0 (control), 5 (RJ5), 10 (RJ10), 20 (RJ20) and 40 (RJ 40) mg/ml. After cooling and freezing-thawing, the percentage of forward progressive motility, viability, abnormality, hypo-osmotic swelling test (HOST) and the mRNA abundance of antioxidant enzymes of spermatozoa were measured. Our results revealed that the addition of 5 mg/ml RJ to the semen extender significantly increased (p < .05) the percentages of forward progressive motility, viability and HOST during liquid and frozen storage. The abnormality of spermatozoa in the RJ5 group was significantly lower compared to the other groups. During liquid storage, a significant decrease in forward progressive motility was found after 48 hr in comparison with 24 hr at 4°C. High levels of RJ (from 10 to 40 mg/ml) were severely decreased the characteristics of rooster spermatozoa in comparison with RJ5 and the control group. The inclusion of RJ at 5 mg/ml to the semen extender enhanced the mRNA transcript of antioxidant enzymes of spermatozoa during liquid preservation. The mRNA abundance of antioxidant enzymes did not influence by cryostorage. Overall, these data suggest that supplementation of RJ at 5 mg/ml to the extender improved semen characteristics and redox status of rooster spermatozoa.

Supplementation of chilling storage medium with glutathione protects rooster sperm quality.

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2-4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2-4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2-4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen.

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.

Effects of k-carrageenan supplementation or in combination with cholesterol-loaded cyclodextrin following freezing-thawing process of rooster spermatozoa.

This experimental research purposely seeks to explore the effect of supplementing k-carrageenan (k-CRG) or CLC (cholesterol-loaded cyclodextrins) or the combined effect of k-CRG and CLC as supplements of antioxidants to an extender for rooster semen freezing. A total of 75 neat pooled ejaculates were collected twice a week from twenty-five (25) commercial line arbor acres broiler roosters (30 wks) during the experimental period. In each replicate, semen samples (n= 15, three ejaculates per rooster) were pooled and divided into nine equal aliquots, and each aliquot was diluted with one of the following extender supplemented with k-CRG, CLC, and k-CRG + CLC after which it was subjected to cryopreservation process using the "pellet" method. In study I, the supplementation of extenders with k-CRG was in five equal aliquots as follows; (0.2, 0.4, 0.6, 0.8) mg/mL and control group (k-CRG 0) mg/mL while in Study II, there was a combination of both k-CRG + CLC (0.4 mg/mL + 1.5 mg/mL, respectively), 0.4 mg/mL k-CRG, 1.5 mg/mL CLC and control group. Sperm quality parameters, endogenous antioxidant enzymes, lipid peroxidation (MDA) and ROS were all assessed after the freeze-thaw process. Our findings in study I indicated that at post-thaw, an optimum 0.4 mg/mL k-CRG supplementation in the extender improved semen quality parameters, endogenous enzymes, MDA and ROS in comparison to the control group. Interestingly prior to the freeze-thaw process, it was depicted in study II that combined k-CRG + CLC (0.4 mg/mL+1.5 mg/mL) inclusion in the extender provided maximum protection to sperm quality parameters, endogenous enzymes, MDA and ROS in comparison to 1.5 mg/mL CLC and control group at post-thaw. Besides, there was also a significant difference observed in the extenders supplemented with combined k-CRG + CLC (0.4 mg/mL +1.5 mg/mL) when compared to 0.4 mg/mL k-CRG for semen quality parameters and endogenous antioxidant enzymes (SOD, CAT, and GPx) but no significant difference was observed for MDA and ROS. Also, there was a significant difference observed in the extender supplemented with 1.5 mg/mL CLC when compared to the control group for semen quality parameters, SOD, CAT, and MDA but no significant difference for GPx and ROS at post-thaw. In conclusion, k-CRG at an optimal dosage of 0.4 mg/mL proved effective for improving post-thaw sperm quality but its combined addition k-CRG + CLC at an optimal concentration of (0.4 + 1.5) mg/mL in the extender provided greater protection to the rooster spermatozoa at post-thaw.

Effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters.

The objective of this study was to evaluate the effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters. Twelve Ross 308 broiler breeder roosters (age: 52 weeks; weight: 4,900 ± 210 g) haphazardly allotted to three dietary treatments (each treatment contained four replicates and one bird in each replicate) for six weeks. Treatments were different levels of flaxseed (0% flaxseed [GFL0], 2% flaxseed [GFL2] and 4% flaxseed [GFL4]). The feed intake quadratically decreased (p < .05) with increasing whole flaxseed levels for the period (58 to 60 weeks). Sperm traits (semen volume and sperm concentration, sperm total and forward motility, sperm viability and morphology, sperm plasma membrane functionality) were evaluated every two weeks (four times), and sperm fatty acid profile was assessed at the end of the experiment. Semen volume, sperm concentration and sperm morphology were not affected by treatments. On week 60, GFL2 group showed a significantly lower percentage of total and progressive sperm motility and sperm membrane functionality in comparison with the control and GFL4 groups. Also, sperm viability was lower in GFL2 group compared with other groups on week 58 (p < .05). In terms of sperm fatty acid profile, GFL2 group significantly reduced the percentage of linoleic acid (C18:2 [n-6]) in comparison with other groups. However, any of the other fatty acids were not affected by dietary flaxseed. In conclusion, dietary supplementation of whole flaxseed could not improve the quality of aged broiler breeder roosters' sperm in this study, nor it could alter the sperm fatty acid profile; thus, it seems necessary to use some antioxidants such as vitamin E in the diet of aged broiler breeder roosters, when supplementing the diets with oils or oilseeds such as flaxseed.

Chicken seminal fluid lacks CD9- and CD44-bearing extracellular vesicles.

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.

Improvement of in vitro proliferation of cockerel spermatogonial stem cells using different combinations of growth factors.

1. This study examined whether in vitro proliferation and maintenance of cockerel spermatogonial stem cells (SSCs) could be improved by adding different combinations of growth factors (GFs), including glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) or leukaemia inhibitory factor (LIF) into the culture medium. 2. The SSCs were isolated from the testes of immature cockerels. For short-term cultures, a medium supplemented with different combinations of GFs for 7 d in 5 replicates was used. The groups were classified as follows: without GF (control group); with GDNF (G group); with GDNF and bFGF (GF group); and with GDNF, bFGF and LIF (GFL group). The number of colonies and cells per colony, as well as the transcript abundance of STRA8 and OCT4 genes, was determined 7 d after the initial culturing. Immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, besides periodic acid-Schiff (PAS) staining, was carried out. 3. The number of colonies and cells per colony increased in the G, GF and GFL groups, compared to the control group (P < 0.01); however, the highest proliferation and colony formation were observed in the GFL group. The positive immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, as well as PAS staining, confirmed the self-renewal and colonisation of cockerel SSCs. The proliferation results were supported by the increased STRA8 and OCT4 transcript abundance in the treated groups (G, GF and GLF), compared to the control group. The SSC proliferation was associated with the higher transcript abundance of STAR8 and OCT4 genes in the GFL group, compared to the G and GF groups (P < 0.01). 4. The results showed that proliferation and colony-forming capacity of cockerel SSCs were positively improved by GDNF, bFGF and LIF. However, the most significant effect was observed when the medium was supplemented with LIF in combination with GDNF and bFGF.

The effect of zinc oxide on rooster semen cryopreservation.

1. Deleterious effects from the freeze-thawing process on post-thawed sperm quality attributes are main limiting factors in cryopreservation. The current study was conducted to determine the effect of semen extender containing zinc oxide (ZnO) on post-thaw rooster sperm quality indices.2. Semen samples from six, 60-week-old broiler breeder roosters were collected weekly during five successive weeks. The samples were mixed and divided into three equal parts and diluted with semen extender containing different levels of ZnO; 0 (ZnO-0), 1 (ZnO-1) or 2 (ZnO-2) µg/ml. After thawing, motility and velocity parameters, plasma membrane functionality, apoptotic like changes, mitochondrial membrane potential (MMP), and DNA fragmentation index (DFI) were evaluated.3. Results showed that the addition of ZnO in the extender quadratically affected (P < 0.01) total motility (TM), progressive motility (PM), and average path velocity (VAP) with the highest values were noted in the ZnO-1 group. Levels of ZnO quadratically affected percentages of live (P < 0.01), apoptotic (P < 0.03) and dead (P < 0.10) spermatozoa, where the highest percentage of live, and the lowest percentage of apoptotic or dead spermatozoa was for the ZnO-1 group. Although adding ZnO quadratically affected plasma membrane functionality and MMP (P < 0.01), it did not affect (P > 0.05) DFI.4. In conclusion, there were some beneficial effects of ZnO supplementation in semen extender on post-thawed rooster sperm quality which may result in a better freezability.