Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Effects of different ultrastructures of lecithin on cryosurvival of goat spermatozoa.

This study was to evaluate the effects of two different ultrastructures of lecithin including nanoparticles (NPE mostly nanomicelles) and lecithin nanoliposome (NLE) with egg yolk extender (EYE) on goat sperm cryopreservation. Semen samples were collected from 6 goats, then pooled, diluted and then frozen. Motility and motion parameters, plasma membrane integrity and functionality, morphology, apoptosis status (Annexin V-PI), acrosome integrity, DNA fragmentation and in vitro fertilisation were assessed. Total motility and most motion parameters were higher in EYE (p < .05) compared with the two lecithin extenders, while there were no significant differences between NLE and NPE. NLE and NPE had higher values for viable spermatozoa (Annexin V-PI) (p < .05) compared with EYE. The highest value for dead spermatozoa was observed in EYE (p = .08). A higher percentage of DNA fragmentation (p < .05) was detected in EYE compared with NPE. Plasma membrane integrity and functionality, morphology, acrosome integrity and fertility of spermatozoa indicated no significant differences between extenders. Data suggested that ultrastructural changes of lecithin (micelles versus. liposome) could not improve the sperm cryosurvival of goat spermatozoa. Moreover, we cannot also claim that lecithin-based diluent supplies better protection compared with the egg yolk in goat.

Beneficial effects of dietary coenzyme Q10 on the productive and reproductive variables of broiler breeder hens.

The aim of this study was to evaluate the effects of supplementary CoQ10 in the diets of aged broiler breeder hens on productive and reproductive variables. A total of 128 hens)44 weeks of age) were randomly assigned to one of 16 groups (eight hens per group). The hen-groups (with equal mean egg production and egg weight) were randomly assigned to one of four diet-groups to provide four pen/groups per treatment. There was no CoQ10 supplementation or supplemental amounts of either 300, 600 or 900 mg CoQ10/kg added to the basal diet. Egg production, weight, and mass were determined weekly. To assess fertility, hatchability, and sperm penetration (SP) rate, the hens were artificially inseminated on a weekly basis (from 47-54 weeks of age). The hens were weighed and killed at the end of the experiment for evaluation of the ovarian morphology, oviduct histology, utero-vaginal junction (UVJ) total antioxidant capacity (TAC), and Pdss2, GDF9, and BMP15 mRNA transcript abundances in the germinal disc regions. The results indicated that there was a linear response curve to increasing amounts of supplemental dietary CoQ10 on fertility, hatchability of eggs, SP rates, TAC of the UVJ, fold height and surface epithelia of the magnum and isthmus, and abundance of GDF9, BMP15 and Pdss2 mRNA transcripts in the germinal disc region. In conclusion, the findings of the present study indicate diet supplementation with CoQ10 had beneficial effects on the productive and reproductive variables of aged hens.

Use of supplemental dietary coenzyme Q10 to improve testicular function and fertilization capacity in aged broiler breeder roosters.

In numerous studies it has been suggested that targeting mitochondria with specific compounds could efficiently inhibit various conditions associated with oxidative stress. The treatment of aged roosters with compounds such as coenzyme Q10 (CoQ10), may improve their reproductive performance by providing protection from oxidative stress. Therefore, this study was performed to assess the effect of supplemental dietary CoQ10 on the testicular function and fertility of aged broiler breeder roosters. A total of 36 roosters)47 weeks of age) were randomly divided into dietary treatments containing either 0, 300 or 600 mg CoQ10/kg diet. Three birds were allocated to each of four replicate groups in each dietary treatment. Between 47 and 54 weeks of age, ejaculates were obtained weekly from the three roosters in each replicate group. Samples in a replicate were pooled and analyzed as a single sample. Between 51 and 54 weeks of age, seminal plasma total antioxidant capacity (TAC), alanine amino transferase (ALAT) and aspartate amino transferase (ASAT) levels were assessed. Fertility, hatchability, and sperm penetration (SP) rates were likewise evaluated. Seminal volume, sperm concentration, sperm plasma membrane functionality, sperm plasma membrane integrity, seminiferous tubule diameter and seminiferous epithelium thickness exhibited quadratic increases in response to increasing levels of dietary CoQ10. Respectively, the 429.19, 433.33, 464.50, 613.50, 392.78 and 447.99 mg/kg dietary concentrations of CoQ10 provided the best results for each of the aforementioned variables. Also, other seminal traits, as well as testosterone concentration, fertility, and SP rates, displayed linear increases in response to the increasing levels of CoQ10. Dietary supplementation of CoQ10 linearly decreased seminal plasma ALAT and ASAT and linearly increased seminal plasma TAC. In conclusion, CoQ10 supplementation in the diet (a minimum of 300 mg CoQ10/kg diet) has the potential to improve the reproductive performance of aged broiler breeder roosters.

Feeding rosemary leaves powder ameliorates rooster age-related subfertility.

Having a high proportion of polyunsaturated fatty acids avian spermatozoa predispose to lipoperoxidation which results in fertility reduction. In the current study, rosemary leaves powder (RLP) was fed to senescent breeder roosters to improve their reproductive performance. Twenty four 70-week-old roosters were randomly divided into four groups and received following treatments including 0 (RLP-0), 2.5 (RLP-2.5), 5 (RLP-5) or 7.5 (RLP-7.5) g of RLP/kg of diet for eight consecutive weeks. Semen characteristics were evaluated weekly. Sperm penetration rate was assessed once, however, fertility, hatchability, embryonic mortality and hatchling quality evaluated twice (using eggs collected during 1st and 2nd weeks following AI) at the end of experiment. Excluding body weight and sperm abnormality percentage, other traits including semen concentration (RLP-2.5 = 3.57, RLP-5 = 4.21 and RLP-7.5 = 3.79; SEM = 0.12; p < 0.01), total sperm production (RLP-2.5 = 1.33, RLP-5 = 1.8 and RLP-7.5 = 1.47; SEM = 0.07; p < 0.01), forward motility (RLP-2.5 = 72.96, RLP-5 = 83.65 and RLP-7.5 = 78.84; SEM = 0.47; p < 0.01) and viability (RLP-2.5 = 82.93, RLP-5 = 88.69 and RLP-7.5 = 86.85; SEM = 0.45; p < 0.01) were improved in RLP treated groups compared to control group (3.08 ± 0.12, 1.05 ± 0.07, 68.39 ± 0.47 and 76 ± 0.45 for semen concentration, total sperm production, sperm forward motility and viability, respectively). In addition, semen volume and sperm plasma membrane functionality were higher in both RLP-5 (0.43 ± 0.01 and 66.66 ± 0.55) and RLP-7.5 (0.39 ± 0.01 and 65.52 ± 0.55) than control group (0.34 ± 0.01; p < 0.05 and 62.89 ± 0.55; p < 0.05). Supplementation of RLP significantly decreased semen Malondialdehyde (MDA) concentration. Moderate level of RLP (RLP-5) had significantly higher numbers of sperm penetration holes compared to other groups. Fertility rate of collected eggs from both RLP-5 (first week: 91.09 ± 1.27 (P < 0.01); second week: 88.73 ± 1.27 (p < 0.05)) and RLP-7.5 (first week: 93.11 ± 1.27 (P < 0.01); second week: 90.87 ± 1.27 (p < 0.05)) groups was higher than other groups at 1st and 2nd weeks of egg collection. Hatchability of eggs set at 2nd week (83.64 ± 3.54; p < 0.05) was higher and embryonic mortality at 1st week (1-6 day mortality: 5.03 ± 1.25 (p < 0.05); 18-21 day and pipped mortality: 8.55 ± 1.31 (p < 0.05)) was in RLP-0.5 group than other groups, respectively. To conclude, RLP supplementation could successfully attenuate age-related sub-fertility in senescent roosters. Further studies are needed to divulge the causal mechanisms involved.

In vitro maturation of ovine oocyte in a modified granulosa cells co-culture system and alpha-tocopherol supplementation: effects on nuclear maturation and cleavage.

This study was designed to investigate the effects of α-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of α-tocopherol (0, 5, 10, 15 µg/ml), oocytes were incubated at 39 °C with 5 % CO2 for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that α-tocopherol had no effect on GVBD and MII as compared to control group, but when α-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm.

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner.