Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen.

The mitochondria-targeted antioxidant MitoQ has been regarded as an effective antioxidant agent against cryo-induced oxidative cellular damage. This study aimed to evaluate the use of different doses of MitoQ combined with trehalose to minimize mitochondrial impairment and oxidative stress during sperm cryopreservation of Markhoz goat. For this, semen samples (n = 50) were collected by electroejaculation every 5 days from 5 bucks in 10 replicates. On each collection day, 5 ejaculates (one ejaculate for each buck) were pooled and then diluted in eight different Tris-based extenders as follows: no additives (control), 20, 200, 2000 nM of MitoQ (MT20, MT200, MT 2000, respectively), 150 mM of trehalose (Tr), MT20+Tr, MT200+Tr, MT2000+Tr. The semen samples were frozen using a standard protocol, and sperm function and oxidative stress were evaluated after thawing. The semen extender supplemented with MT200+Tr had higher (P < 0.05) total and progressive motility, acrosome and membrane integrity, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and lower (P < 0.05) DNA fragmentation, malondialdehyde and intracellular hydrogen peroxide levels than the all other groups except MT200; meanwhile, MT200 was also improved (P < 0.05) in these parameters than in the control group. Furthermore, MT200 and MT200+Tr showed higher (P < 0.05) percentages of live cryopreserved sperm with high mitochondrial activity than other groups. However, abnormality percentage and catalase activity of frozen-thawed sperm were not affected by treatments (P > 0.05). To conclude, we have found that supplementation of 200 nM MitoQ alone or in combination with 150 mM trehalose to semen extender improved the quality of cryopreserved sperm in goats, which is associated with enhanced antioxidant enzymatic defense and mitochondrial activity and reduced DNA fragmentation.

Protective effects of Tribulus terrestris and Cinnamomum zeylanicum extracts and trehalose added to diluents on goat epididymal sperm freezability.

This study investigates the effect of adding Tribulus terrestris ethanol extract (TEE) and Cinnamomum zeylanicum ethanol extract (CEE) and trehalose on freezability of goat epididymal spermatozoa. In Experiment 1, the treatments consist of basic extender containing 25, 50 or 100 µg/ml of TEE or CEE. The control contained no additives. Experiment 2 was carried out to compare the effect of best concentrations resulted in the first experiment with 150 mM trehalose added to basic extender. The results of experiment 1 showed that supplementation of 50 µg/ml TEE and 50 µg/ml CEE increased significantly the percentages of motility, progressive motility and viability of cryopreserved spermatozoa, while the level of malondialdehyde concentration was decreased. Moreover, the 50 µg/ml TEE treatment indicate significantly) P < 0.05) the lowest DNA fragmentation among the other treatments. The data obtained from experiment 2 show that all treatments increased significantly) P < 0.05) the percentages of total motility, viability and membrane integrity, and concurrently decreased the rate of MDA compared to control. In addition, the rates of viability and progressive motility were significantly (P < 0.05) higher in diluents contained herb extracts and trehalose. Regarding DNA fragmentation, the results demonstrate that using the extracts and trehalose in diluents decreased the DNA damages and thereby improved the rate of intact sperm heads. In conclusion, the results of this study indicate that 50 µg/ml of Tribulus terrestris and Cinnamomum zeylanicum ethanolic extracts alone and plus trehalose improved the spermatozoa quality and could be used for cryopreservation.

Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa.

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7-10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa.

Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm.

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 109 sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 µg/ml of Purslane aqueous extract (PAE25µg/ml, PAE50µg/ml, PAE100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane methanolic extract (PME25µg/ml, PME50µg/ml, PME100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane ethanolic extract (PEE25µg/ml, PEE50µg/ml, PEE100µg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE50µg/ml, PME50µg/ml and PEE50µg/ml than those of the control. In addition, PME50µg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME50µg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME50µg/ml and PAE50µg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME50µg/ml and PAE50µg/ml treatments. In conclusion, the results of this study indicated that 50 µg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.

Evaluation of pentoxifylline and Basal Medium Eagle supplemented to diluent on cryopreserved goat spermatozoa.

This study investigated the effect of pentoxifylline (PTX) and Basal Medium Eagle (BME) on frozen-thawed goat spermatozoa. Immediately after initial examination of ejaculated semen, samples were pooled and reexamined for quality. Then, samples were divided into eight equal aliquots and diluted with a basic tris-extender containing PTX (3, 6, 9 mM) and BME (5 mM) to reach a final concentration of 25 × 109 and frozen. After 24 hr, the samples were individually thawed at 37°C for 30 s and evaluated for different characteristics. Obtained post-thaw results from Computer-Assisted Sperm Analysis indicate using of 3 and 6 mM PTX led significantly to an improvement in total motility, progressive motility and velocity characteristics of spermatozoa, except the beat/cross frequency (BCF) which indicated statistically no differences (p > .05) among control and treatments. Diluents prepared with BME (5 mM) and PTX alone (3 and 6 mM) improved significantly the membrane integrity-functionality, acrosome integrity and also hyaluronidase activity. Regarding recovery rate, the results showed significantly (p < .05) higher values for diluents containing 3 and 6 mM PTX compared to other groups. Malondialdehyde concentration exhibited also a significant difference (p < .05) in diluents supplemented with 5 mM BME, 3, 6 and 9 mM PTX, and mixture of 3 mM PTX and 5 mM BME which illustrate a similarity for active mitochondria, apoptotic-like and dead spermatozoa. Finally, the ratio of sperm chromatin dispersion stained spermatozoa presented significant differences (p < .05) among treatments in which the diluents added PTX alone demonstrated significantly lower values than control and extenders containing the mixtures of BME and PTX. In conclusion, the observation in this study indicates using of 3 and 6 mM PTX and BME alone may improve significantly (p < .05) the quality of cryopreserved goat spermatozoa.

The Influence of Male Biostimulation on Cloacal Anatomy and Egg-Laying Behavior in Young Female Muscovy Ducks (Cairina moschata forma domestica).

The importance of Muscovy ducks in industrial poultry production is growing; however, little is known about the physiology of their reproductive cycles. This study investigated the influence of male biostimulation on female ducks before the commencement of the laying phase. A total of 30 muscovy ducks, hatched in the same year at 289-341 days of age, were divided into two groups of 15 birds each and kept with and without contact with a male duck until the day of first egg-laying-319 ± 14 and 335 ± 13, respectively. Before reaching egg-laying maturity, the cloacae of 29 adult ducks were subjected to daily clinical assessments. The evaluations yielded four unique categories of outcomes, determined by assessing factors such as the degree of redness and protrusion of the mucous membrane, the moisture level, and swelling of the cloacal sphincter muscle. The results of this study on biostimulation revealed that, on average, female ducks that had contact with males laid their first egg 16 days earlier, weighing 78.7 ± 3.0 g, compared to the isolated female ducks, weighing 79.1 ± 7.0 g. Furthermore, there was no significant difference observed in the mean initial egg weight between the groups (p = 0.841). The cloacal morphology indicated significant morphological changes 25-26 days before laying. Efforts to improve Muscovy production and develop biotechnological techniques to modify these ducks' reproductive cycle will benefit from these advancements.

Supplementation of Rooster Semen Extender with Aqueous Extract of Urtica dioica for a Long Time Preservation by Low Temperature.

The peroxidation of spermatozoa membrane phospholipids is a primary cause of irreversible changes in the preservation of avian semen. To address this issue, the objective of the present study was to assess the potential of Urtica dioica extracts in protecting avian spermatozoa during prolonged storage. Gas chromatography-mass spectroscopic techniques were employed to evaluate the bioactive compounds present in the aqueous and ethanolic extracts obtained from the aerial parts and roots of U. dioica. Semen samples were collected from 16 roosters twice a week and were diluted in Lake's extender containing different concentrations (0, 0.5, and 1 mg/100 mL) of the various extracts. Subsequently, the extended semen samples were cooled and stored at 5°C, and the sperm quality parameters were assessed at 0, 12, 24, and 36 hours of storage. The data from this experiment clearly demonstrate that the addition of nettle root aqueous extracts to the semen diluent, especially at a concentration of 0.5 mg/100 mL, resulted in a significant improvement in various sperm quality parameters. Notably, there were enhancements in total and progressive sperm motilities, viability, fertility, membrane integrity, acrosomal membrane integrity, and a reduction in malondialdehyde production in rooster semen stored in vitro for up to 36 hours. Interestingly, the present study reveals that the beneficial effects of the aqueous extracts from different parts of the nettle were supported not only by the conventional manual method but also by the computer-assisted sperm analysis system. This dual confirmation further emphasizes the positive impact of the aqueous extract on various sperm traits during cooled semen preservation. In conclusion, this study highlights the potential of U. dioica extracts, particularly the aqueous extract from nettle roots at a concentration of 0.5 mg/100 mL, in safeguarding avian spermatozoa during prolonged storage. The significant improvements in various sperm quality parameters and the validation of results through both manual and computer-assisted analysis methods provide strong evidence for the application of U. dioica extracts in avian breeding programs and artificial insemination practices.

Evaluation of Ethanolic Extract of Prosopis Farcta and Trehalose on Cryopreserved Goat Epididymal Spermatozoa.

The purpose of this study, carried out in two experiments, was to investigate the antioxidant effect of Prosopis farcta in Experiment 1, and trehalose in Experiment 2 in the freezing extender, on the quality of frozen-thawed goat epididymal spermatozoa. Sperm samples were added to based egg-yolk Tris-extender containing experimental treatments. The first experimental treatments included the following: an extender of the control group without additive and extender containing 50, 100, or 150 µg/mL of Prosopis farcta ethanolic extract (PEE1, PEE2, and PEE3, respectively). Treatments of the second experiment include an extender of the control group without additive, an extender containing 100 mM of trehalose (Tr), an extender containing 100 µg/mL PEE2, and 100 µg/mL PEE2 + 100 mM Tr. The results of the first experiment showed that PEE2 compared with the control group led to a significant decrease (p < 0.05) in malondialdehyde (MDA) concentration. Also, PEE1 and PEE2 treatments resulted in a significant increase (p < 0.05) in motility parameters by computer-assisted sperm analysis, and MDA concentrations decreased significantly (p < 0.05) in all treatments compared with the control group. In general, the results of the present experiment showed that Prosopis farcta ethanolic extract at the level of 100 µg/mL was effective in improving the quality of frozen-thawed goat epididymal spermatozoa. Also, a combination of Prosopis farcta ethanolic extract and trehalose can be successful in freezing goat epididymal spermatozoa.

Effects of Antifreeze Protein Type I and Glycerol in Diluents on Cryopreserved Goat Epididymal Sperm.

The effect of antifreeze protein (AFP) as a cryoprotectant used in different concentrations of glycerol on post-thaw quality of epididymal sperm was investigated. Sperm were isolated from 50 testicles, obtained from 25 healthy mature goat bucks, with progressive motility >80%, and total morphological abnormalities <10% were pooled in each replication. The semen samples were diluted with Tris-citrate-fructose-soybean lecithin extender containing different concentration of AFP [0 µg/mL (A0), 5 µg/mL (A5), 10 µg/mL (A10)]. Each concentration of AFP was added in an extender containing either 7% (G7) or 5% (G5) glycerol. Post-thaw total and progressive motility were found to be higher (p < 0.05) in groups A5G5 and A5G7. Plasma membrane integrity, sperm acrosome integrity, DNA integrity, acrosome intact sperm, and mitochondrial membrane potential were found to be higher (p < 0.05) in groups A5G5 and A10G5. Sperm viability was found to be higher (p < 0.05) in group A5G5, while lipid peroxidation was recorded lower (p < 0.05) in groups A5G5 and A5G7. Regarding the apoptosis occurrence, the results demonstrate higher (p < 0.05) live post-thawed spermatozoa for groups containing 5 µg/mL AFP with 5% and 7% glycerol in addition to the lowest (p < 0.05) value for groups containing 0 µg/mL AFP with 5% and 7% glycerol. Based on these results, the present study concludes that the addition of 5 µg/mL AFP in combination with 5% glycerol in freezing extender improves the post-thaw quality, structure, and function parameters for buck spermatozoa.

Use of Purslane (Portulaca oleracea) Extracts and Cysteine in Diluents to Improve the Cryopreserved Quality of Goat Sperm.

Highlights Using cysteine and purslane extracts in extenders improved significantly the post-thaw sperm characteristics. Sperm viability, DNA integrity, and mitochondrial activity demonstrate an improvement in post-thaw sperm. Malondialdehyde production was decreased based on the positive effects of treated extenders. The obtained results demonstrate that supplementation of 50 µg/mL of purslane methanolic extract with cysteine to freezing extenders was significantly superior compared with other treatments.

Letrozole, an aromatase inhibitor, improves seminal parameters and hormonal profile in aged endangered Markhoz bucks.

OBJECTIVE: Letrozole, a potent aromatase inhibitor, is known to have the potential to modify male reproductive function by altering sex hormone levels. This study aimed to evaluate the semen and testicular characteristics and hormonal profile of aged Mrakhoz bucks (Capra hircus) treated with letrozole. METHODS: Twelve Markhoz male goats, aged between 4.5 to 5.5 years with an average body weight (BW) of 61.05±4.97 kg were used for the study. Animals were randomly divided into two equal groups and subcutaneously received either 0.25 mg/kg BW of letrozole or a control every week for 2 months. The semen collections were performed every 10 days, and blood samples and testicular biometric records were collected at 20 days intervals. RESULTS: Letrozole causes increased testosterone and follicle-stimulating hormone levels, testosterone to estradiol ratio, semen index and reaction time during the period from 20th to 60th days (p<0.05). Furthermore, letrozole-treated bucks had higher semen volume, sperm concentration, and total sperm per ejaculate from 30th to 60th days (p<0.05). However, no differences occurred between the groups in scrotal circumference, relative testicular volume, semen pH, abnormality, acrosome integrity, and membrane integrity of sperm during the study (p>0.05). The serum luteinizing hormone levels, sperm viability, motility, and progressive motility increased, and estradiol levels decreased after 40th to 60th days of letrozole treatment (p<0.05). CONCLUSION: Letrozole application to aged Markhoz bucks provokes reproductive hormonal axis which, in turn, induces enhancement of semen production and quality.

Protective Effects of Trehalose and Pentoxifylline on Goat Sperm Exposed to Chilling-Freezing Process.

Freezing of sperm is known as an important part of assisted reproduction. However, many studies have illustrated that cryopreservation negatively affects the quality and fertility rate of sperm. This study aimed to evaluate the effects of trehalose and pentoxifylline (PTX) in diluents on cooled and frozen-thawed Markhoz goat sperm. Preassessed samples were pooled and diluted with a basic diluent using trehalose and PTX. The cooled sperm showed significant improvement. The motion characteristics of cryopreserved sperm were evaluated based on computer-assisted system analysis. In this study, we investigated the viability, membrane integrity, malondialdehyde concentration, total abnormality, acrosome integrity, and seminal hyaluronidase enzyme. Also, the hypo-osmotic swelling test, mitochondrial activity, apoptotic features, caspase activity, chromatin dispersion test, active mitochondria, and reactive oxygen species (ROS) activity were assessed as complementary parameters. The data illustrate that the total motility, progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and the ratio of sperm chromatin dispersion, viable sperm were improved significantly (p < 0.05) using 3 mM PTX alone or 3 mM PTX plus 50 mM trehalose, while other characteristics indicate significant enhancement by 3 and 6 mM PTX and 50 and 70 mM trehalose alone or in combination, except amplitude of lateral head displacement (ALH), beat/cross frequency (BCF), and intracellular ROS-(O-), which demonstrate no significant difference among treatments. In conclusion, this study indicates that addition of 3 and 6 mM PTX alone or with 50 and 70 mM trehalose seems to reduce the damage caused by cooling and cryopreservation processes.

Effect of orally administrated letrozole on reproduction performance and gene expression of FOXJ1, LPR2 and PVRL3 in reproductive tract in aged roosters.

letrozole is an aromatase inhibitor that stops the production of estrogen through interrupting the entrance of hormone androgen into a small amount of estrogen. Therefore, the current study was developed to estimate orally administrated Letrozole on the reproductive performance and relative abundance of Foxj1, PVRL3, and LPR2 mRNA in aged roosters. Fifty-five-week old ROSS 308 breeder roosters (n = 18) were orally treated using letrozole. Primarily, the body weight of the animals was recorded, and they were randomly classified into three groups (n = 6 birds/group) receiving different doses of Letrozole, including 0, 0.015, and 0.03 mg/kg body weight/day for three weeks. At the end of the trial, seminal traits, plasma, testicular hormone levels (testosterone, estradiol, and FSH), histopathological studies, in vitro fertility, and relative abundance of testis PVRL3, epidydimal Foxj1, and LPR2 mRNA were evaluated. Based on the results, the sperm quality variables were statistically higher in the 0.03 group compared to the controls. Greater histologic parameters, such as diameter of seminiferous tubules, thickness of seminiferous epithelium, categorized epididymal region, and in vitro fertility rates were estimated for the treated groups(p < 0.001). Plasma and testicular testosterone, estradiol concentrations, and plasma FSH levels were significantly influenced by letrozll treatment (p < 0.001). Relative mRNA transcript abundance increased for PVRL3 and decreased for Foxj1 and LPR2 in treated groups. Overall, aromatase inhibitors can enhance the reproductive performance of aged commercial broiler breeder roosters. However, it can impact endocytosis and ciliogenesis events via reducing estradiol.

Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa.

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. The aim of this study was to investigate the effects of different concentrations of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on the cryopreservation of goat spermatozoa in tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with TCG, the second aliquot was mixed with TCG and egg yolk (TCGY), third aliquot was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquots were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3mg/ml CLC. All samples were cryopreserved in straws over liquid nitrogen vapor and sperm motion Kinetics were measured by computer-assisted semen analysis (CASA) (percent motility (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH)). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant (p<0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thawed motility, progressive motility and recovery rate were significantly (p<0.05) higher in 1.5mg/ml CLC with 2.5% BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome (P>0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing.

Effects of letrozole administration on growth and reproductive performance in Markhoz goat bucklings.

This study evaluated the growth performance, testicular and semen characteristics, and hormonal profile of Markhoz (Iranian Angora) bucklings injected with letrozole (LTZ). Twenty-eight 4-4.5 month old bucks were randomly assigned into four groups and received either 0.25 mg/kg body weight (BW) LTZ subcutaneously (sc LTZ) or intramuscularly (im LTZ), and also sc (sc CONT) or im (im CONT) controls every week for 3 months. The study was performed at the beginning of the breeding season in Sanandaj Animal Husbandry Research Station (46.99 °E, 35.31 °N). The results showed that LTZ causes increased final body weight (25.78 ± 1.61 kg), higher average daily gain (104 ± 0.03 g/days), and decreased feed conversion ratio (7.81 ± 2.57) (P < 0.05). The pre-slaughter, hot, and cold carcass weights (27.56 ± 2.40, 11.45 ± 1.07 and 11.11 ± 1.05 kg, respectively) were (P < 0.05) heavier in LTZ groups while other carcass characteristics did not differ between groups. No differences occurred between the groups in biochemical parameters, except high-density lipoprotein levels (35.47 ± 2.43 mg/dL) which was higher in LTZ treatments (P < 0.05). LTZ-treated bucks had larger scrotal circumference (20.12 ± 5.75 cm), higher relative testicular weight (560.91 ± 78.59 mg/100 g BW) and volume (175.5 ± 29.71 cm3), greater diameter of seminiferous tubules (224.5 ± 5.21 µm), and number of Sertoli cells (8.39 ± 0.77) (P < 0.05). Semen volume (0.74 ± 0.16 mL), sperm concentration (2.64 ± 0.19 × 10-9/mL), total sperm per ejaculate (1.95 ± 0.49 × 10-9), and semen index (1248 ± 323) increased (P < 0.05) by LTZ treatments, while semen pH (6.77), motility (80.91%), progressive motility (76.75%), viability (83.35%), abnormality (13.70%), acrosome integrity (78.06%), and membrane integrity (80.05%) of sperm remained unaffected. Intratesticular and serum testosterone (T) levels (7.97 ± 0.89 ng/mg protein and 2.47 ± 0.59 ng/mL, respectively), serum luteinizing hormone (LH), growth hormone (GH) levels (1.71 ± 0.24 and 3.62 ± 0.33 ng/mL, respectively) of LTZ groups were elevated, whereas intratesticular and serum estradiol (E2) levels (84.14 ± 8.15 pg/mg protein and 32.33 ± 2.16 pg/mL, respectively) decreased (P < 0.05). No differences were recorded between the sc and im routes of LTZ administration in the measured parameters. To conclude, we have found that LTZ treatment improves growth and reproductive functions of goat bucklings associated with increased serum LH and GH, elevated T and reduced E2 levels in both serum and testis.

Anti-apoptotic effects of minocycline on ram epididymal spermatozoa exposed to oxidative stress.

Cryopreservation causes major damage to sperm cells and thereby, increased apoptosis has been documented as physiologically programmed cells death. The goal of current study was to evaluate the anti-apoptotic effects of minocycline compared to sericin added to diluents on frozen-thawed spermatozoa. Epididymal spermatozoa isolated from 50 pairs testes with motility >80% and total morphological abnormalities <10% were pooled, divided into 9 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing minocycline (5 and 10 mg) and tert-butyl hydro peroxide (10 and 20 µm) and sericin (0.5 gr). Total sperm motility and progressive motility were evaluated by Computed Assisted System Analysis (CASA). Acrosome and plasma integrity, viability, hypo osmotic swelling test, malondialdehyde concentration, DNA fragmentation test, mitochondrial activity, apoptotic features, caspase activity and level of H2O2 and O2 were assessed as completed characteristics of frozen-thawed spermatozoa. The results indicate that 10 mg minocycline added to extender resulted in significant (P < 0.05) enhancement of total motility, progressive motility and viability of post-thawing spermatozoa. The results of plasma membrane demonstrate significantly (P < 0.05) higher value of both minocycline concentrations with 10 µm tert-butyl hydro peroxide. In regard to caspase activity, diluents supplemented with 5 and 10 mg minocycline improved significantly (P < 0.05) the rate of viable spermatozoa. The results of malondialdehyde concentration show diluents supplemented with 10 mg minocycline and 0.5 gr sericin were significantly (P < 0.05) lower than other extenders. The ratio of sperm chromatin dispersion stained spermatozoa illustrated the higher rate in extenders containing 10 mg minocycline plus 10 µm tert-butyl hydro peroxide, 5 mg minocycline plus 10 µm tert-butyl hydro peroxide and 5 and 10 mg minocycline were significantly (P < 0.05) than other treatments. Moreover, the results of mitochondrial activity show significantly (P < 0.05) an improvement by supplementation of 5 and 10 mg minocycline. Regarding the apoptosis occurrence and intracellular ROS, the results demonstrate significantly (P < 0.05) higher live post thawed spermatozoa and lowest value for diluents containing 10 and 5 mg, respectively. In conclusion, the results indicate that addition of 10 mg minocycline seems to reduce the apoptotic marker during cryopreservation of epididymal ram spermatozoa.

The effects of sericin on cryopreserved sperm cells and subsequent embryo development in mice.

BACKGROUND: Sericin, because of its ability to remove free radicals and its antioxidant properties, has been used to successfully cryopreserve various mammalian cell types. However, the effects of sericin on cryopreservation of mouse sperm has not been reported. OBJECTIVE: The current study intended to determine the protective role of different concentrations of sericin (0, 0.25, 0.5, and 0.75%) on mouse spermatozoa during cryopreservation, in addition to its effect on in vitro fertilization and subsequent embryo development. MATERIALS AND METHODS: Mouse sperm from epididymides were frozen in cryoprotective agent with 18% raffinose, 3% skim milk, and different concentrations of sericin (0, 0.25, 0.5, 0.75%). Thawed sperm were used for in vitro fertilization. The obtained embryos were cultured in Ksom medium for 6 days. The post-thawed motility, viability, fertilizing ability, and subsequent development to the 2-cell embryo and blastocyst stages were evaluated. RESULTS: Our findings show that frozen-thawed sperm cells with 5% sericin indicate significantly (p≤0.0001) percentages of survivability and motility, the best fertilizing ability, as well as 2-cell embryo and blastocyst development compared to the other treated groups. There was no significant difference in survivability (p=0.8781), fertilizing ability (p=0.2458) and development of 2-cell (p=0.5136) and blastocysts embryos (p=0.0896) between 0.75% sericin and control groups. CONCLUSION: Supplementation by 0.5% sericin in cryoprotective agent improved frozen-thawed mouse epididymal sperm cell quality and resulted in increased embryo development.