Cryopreservation of ram semen: baicalein efficiency on oxidative stress, chromatin integrity, viability and motility post thaw.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.

Investigation of the effects of syringic acid supplemented to Tris semen diluent on ram semen freezability.

To the best of our knowledge, no research has been conducted to test the effects of syringic acid (SA) on ram semen freezing within the scope of natural antioxidants added to semen extenders. Therefore, this study had two main objectives. First, to test whether adding SA to ram semen freezing extender has a protective effect and contributes positively to sperm kinetic, plasma and acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidant and antioxidant and DNA damage parameters after thawing. Second, it was to determine at what concentration the SA supplemented to the extender could be applied by in vitro studies by preserving the fertilization ability of frozen semen at the highest level. In the study, six individuals of Sönmez rams were used. The semen was collected from the rams using an artificial vagina and pooled. The pooled semen was divided into five different groups and extended with 0, 0.5, 1, 2 and 4 mM SA (control C, SA0.5, SA1, SA2 and SA4, respectively). After dilution, the semen samples were kept at 4°C for 3 h, then loaded into 0.25 mL straws and frozen in liquid nitrogen vapour. The SA1 and SA2 groups were higher plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), plasma membrane integrity and motility compared to other groups (p < .05). It was observed that SA supplemented to the Tris extender significantly reduced DNA damage, and the lowest values were obtained especially in the SA1 and SA2 treatments (p < .05). Also, lowest MDA level was determined at the SA1 and this was statistically significant compared to SA4 and C (p < .05). In conclusion, it was revealed that SA added to Tris semen extender at 1 and 2 mM treatment doses increased progressive and total motility and preserved PMAI, plasma membrane integrity, HMMP and DNA integrity.

The interaction between seminal plasma biochemical constituents and short-term semen preservation in Honamli and hair goats.

This study investigated the short-term storage of Honamli and Hair buck semen based on the relationships among the biochemical constituents of the seminal plasma. In total, 12 Honamli and Hair goat bucks (six of each breed) were used. Collected individual semen samples were divided into two parts, one placed in short-term storage and the other centrifuged to determine the biochemical constituents of the seminal plasma. Diluted semen samples were cooled to +4°C; the samples were stored at this temperature; the spermatological parameters (i.e., motility, plasma membrane and acrosome integrity [PMAI], viability and high mitochondrial membrane potential) were assessed at 0, 24, 48, 72 and 144 h. A significant positive correlation was determined between the PMAI, motility and viability with globulin, chlorine, alanine aminotransferase (AST) and aspartate aminotransferase (ALT). A positive correlation was detected between the PMAI and viability with total protein. In contrast, a negative correlation was determined between the motility and viability with sodium values in buck semen (p Ë‚ .05). Also, it was evaluated the Hair buck samples became inviable across all parameters at 72 h, while the Honamli buck samples continued to be viable at 144 h. In conclusion, the seminal plasma biochemical parameters of total protein chlorine, globulin, albumin, sodium, AST and ALT correlated with spermatological parameters in buck semen. The factors of animal (p Ë‚ .05) and time (p Ë‚ .001) affected the spermatological parameters, but there was no interaction between the two factors (p > .05) during the short-term storage period. Also, it was determined that Honamli buck semen was more resistant to cold injury than Hair buck semen against cold injury during storage.

Investigation of Changes in Spermatozoon Characteristics, Chromatin Structure, and Antioxidant/Oxidant Parameters after Freeze-Thawing of Hesperidin (Vitamin P) Doses Added to Ram Semen.

We conducted this study to determine the potential cryopreservative effects of different hesperidin (vitamin P; H) doses on ram semen after freeze-thawing. Semen samples were obtained from Sönmez rams using an artificial vagina. The samples were divided into six groups: control, 10, 50, 100, 250, and 500 µg/mL H (C, H10, H50, H100, H250, and H500, respectively). At the end of the study, sperm motility and kinetic parameters, acrosome integrity (AI), mitochondrial membrane potential (MMP), viability, lipid peroxidation levels (LPL), chromatin damage, oxidant parameters, and antioxidant parameters were assayed. None of the doses of H added to the semen extender showed any enhancing effects on progressive motility compared to C (p > 0.05). In fact, H500 had negative effects (p < 0.05). Moreover, AI was the highest at the H10 dose, while LPL values were the lowest at the same dose (p < 0.05). The doses of H10 and H50 added to the Tris extender medium showed positive effects on sperm cell chromatin damage. Consequently, we can say that H doses used in this study are not effective on semen progressive motility, but the H10 dose is effective on AI and chromatin damage by reducing LPL.

Thymoquinone improves motility, plasma membrane integrity and DNA integrity of frozen-thawed ram semen.

The aim of this study was to determine the effects of thymoquinone (TQ), which is the most essential active compound of Nigella sativa, on the spermatological parameters of ram semen during cryopreservation. Ejaculates were collected from five Sonmez rams using an artificial vagina and extended with Tris-based extender not containing TQ (control, 0 µg/ml TQ) and containing 10, 25, 50 and 100 µg/ml TQ. The extended semen samples were equilibrated in a + 4°C cold cabinet for 2 h. After 2 h, the samples were loaded into 0.25 ml French straws. The straws were frozen by liquid nitrogen vapour and stored in a liquid nitrogen container (-196°C). The frozen straws were thawed in a water bath (37°C for 30 s) and evaluated in terms of motility characteristics, plasma membrane and acrosome integrity, mitochondrial reactive oxygen species levels, lipid peroxidation levels, DNA damage and biochemical alterations (oxidative stress index, malondialdehyde and glutathione). TQ100 had higher total motility (53.59 ± 3.01) and progressive motility (19.84 ± 1.44; not significantly different from TQ25 and TQ50) compared to the control and TQ10 (p Ë‚ 0.05). According to the results of the analyses on motility characteristics, there were significant differences between the groups in terms of curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and linearity (LIN; p Ë‚ 0.05). The highest DNA damage was detected in the control group (p Ë‚ 0.05). TQ50 had higher plasma membrane and acrosome integrity (59.56 ± 5.92) compared to the control and TQ25 (p < 0.05) but not significantly different from TQ10 and TQ100. The lowest mitochondrial reactive oxygen species levels were detected in TQ50 and TQ100 (p Ë‚ 0.05). There were no significant differences among the groups in terms of their oxidative stress index, lipid peroxidation, malondialdehyde and glutathione levels (p > 0.05). According to the results, it could be concluded that supplementing 50 or 100 µg/ml TQ to Tris extenders that were used for ram semen cryopreservation showed a positive effect on motility, plasma membrane integrity and acrosome integrity, and it reduced DNA damage and mitochondrial reactive oxygen species levels.

Protective role of the dried white mulberry extract on the reproductive damage and fertility in rats treated with carmustine.

The present study investigated the protective effect of dried white Mulberry extract (DWME) against carmustine (Crm) induced biochemical alterations and spermatological, histopathological, and fertility damage in Wistar albino rats. Male rats were divided into four groups (control, Crm, Crm + DWME, and DWME group). It was found that Crm decreased the motility. Crm decreased the concentration (not different from control group) compared to DWME groups. Total blood MDA levels were reduced during the recovery period. Also, the recovery period reduced the MDA levels in the Crm group/testicular tissue. The GSH levels in the Crm + DWME group were the highest among all groups in the testicular tissue/experiment period. In the immunohistochemical evaluation of the testicular tissue, a high level of caspase-3 was observed in the cells that underwent meiosis in the Crm group. The most pronounced DNA damage was also detected in the Crm group. The Crm + DWME group showed the highest number of offspring born during recovery period. In conclusion, dried white mulberry extract protects against the spermatological damages caused by carmustine. Moreover, recovery period played a positive effect on spermatological parameters and fertility.

Use of Infrared Thermography during Ejaculation Process and Its Link with Semen Quality and Freezability in Dogs.

This study aimed to describe the thermal variation of external reproductive tracts during ejaculation in relation to sperm quality in dogs. Forty-six adult fertile dogs were monitored using a thermal camera before, during and after the semen collection, taking into account penile and scrotal temperatures as reproductive thermal patterns while eye and perianal temperatures were recorded as complementary thermal patterns of behavioral response. The parameters were classified depending on age (≤4 years and >4 years), body weight (BW) (≤75 kg and >75 kg), sperm concentration (CON) (≤300 million and >300 million), total testicular volume (TTV) (≤600 cm3 and >600 cm3) and total ejaculation time (TET) (≤800 s and >800 s) of the animals from which semen was collected successfully. Heavier males (p < 0.05) that have more consistent testicles (p < 0.01) as well as quicker ejaculate responders (p < 0.001) and lower scrotal temperature had better semen (Δ motility) freezability. The lower eye temperature prior to the ejaculation (p < 0.01), lower scrotal temperature following ejaculation (p < 0.01), and conversely, higher penile temperature during the ejaculation (p < 0.001) had a higher sperm concentration. Furthermore, the sperm freezability was negatively correlated with total ejaculation time (r = -0.39, p < 0.05) and sperm abnormalities were lower in the ejaculate of dogs having a higher temperature of the scrotum, bulbus and penis. In conclusion, infrared monitoring throughout semen collection in dogs can provide information on behavioral reactions during human manipulation, as well as semen quality and testicular functionality.

Gallic acid improves the viability and mitochondrial membrane potential of post-thawed goat buck semen.

The aim of this study was to determine the effects of gallic acid (GA) on frozen-thawed goat spermatozoa. Four Honamli goat bucks were used at their breeding season, and ejaculates were collected by an electroejaculator. Mixed semen was divided into the following four groups: control (0 mM), low (L; 1 mM), medium (M; 2 mM), and high (H; 4 mM) concentration of GA. All the groups were frozen and thawed in a water bath for spermatological evaluation. The lowest motility was observed in the control group (47.60 ± 5.70%) (P < 0.05), while the highest viability (62.45 ± 1.68%), plasma membrane and acrosome integrity (44.81 ± 4.57%), and high mitochondrial membrane potential (35.96 ± 2.50%) were observed in the low GA group (P < 0.05). Also, the lowest hypo-osmotic swelling test (HOS +) value was found in the high GA group (47.60 ± 4.82%) (P < 0.05). In conclusion, supplementing a low concentration (1 mM) of GA to the Tris-based semen extender had a positive effect on spermatological parameters after freeze-thawing of Honamli goat semen. Further studies should be continued in other species with different doses and combinations using commercial and/or homemade semen extenders.

Red pine (Pinus brutia Ten) bark tree extract preserves sperm quality by reducing oxidative stress and preventing chromatin damage.

This study aimed to investigate the effectiveness of using red pine bark tree extract (P; Pinus brutia Ten) as a TRIS extender in an attempt to prevent oxidative stress in bull spermatozoa after freezing. Semen specimens were obtained from Simmental bulls via an artificial vagina and pooled. They were separated into five specimens and diluted with Tris extender consisting of P (200, 100, 50 and 25 µg/ml) and P free (control; C) up to a final concentration of 16 × 106 per straw. All specimens were equilibrated for a period of 4 hr at a temperature of 4°C, following which they were filled in 0.25-ml French straws and frozen. Addition of P resulted in favourable tail length in comparison with C (p < .05). The lowest malondialdehyde levels and the highest glutathione levels were detected in all P groups (p < .05). Supplementation with P did not show advanced results in terms of total, progressive sperm motility and total abnormality in comparison with C (p > .05). In conclusion, it has been shown that although P added to a Tris extender does not have a positive effect on sperm motility, it prevents chromatin damage by reducing oxidative stress, in addition to reducing head abnormalities when used at the amount of 50 µg/ml.

Genomewide association of male reproductive traits in Aksaray Malakli dogs.

Male reproductive parameters are often used for the functional examination and evaluation of predicted genetic values for future aspects. However, these traits are relatively reliable until the measurable effects are expressed on desired traits. Therefore, we aimed to associate the single nucleotide polymorphism (SNP) genotype of the investigated characteristics and reproductive loci. A total of 46 male dogs are divided into three age groups (I ≤ 3 years, n = 19; II 4-6 years; n = 19, and III ≥7 years, n = 8). The testis, scrotum and body weight, libido sexualis and ejaculation time for each fraction were monitored as functional traits, while the pH, fractional semen volume, motility, concentration, and abnormal and dead spermatozoa rate were recorded as spermatological traits. The Affymetrix Canine 127 K SNP genotyping array v2 (Affymetrix Inc., California, USA) was used for SNP genotyping. In the primary results, the scrotal circumference was found to be higher in group II compared to other groups (p < 0.05) and the lowest total abnormal spermatozoa rate was found in group I (p < 0.05). The normal spermatozoa rate was found to be significantly above the threshold in relation to the SNP in chromosome 17. In conclusion, this study represents an exciting first step towards SNP association with dog semen spermatological parameters. Future studies might be undertaken to evaluate this SNP region for gene-knockout and expression analysis and for fine mapping to validate and/or discover the exact position of the effect region.

Effect of cholesterol loaded cyclodextrin on semen cryopreservation of Aksaray Malakli shepherd dogs of different ages.

The objective of the study was to determine the effect of cholesterol-loaded cyclodextrin (CLC) on the quality parameters of semen from Aksaray Malakli Shepherd dogs of different age groups. Forty-eight male dogs were divided into 3 groupings according to their ages (young age (Y): ≤3 years, n: 20; middle age (M): 4-6 years, n: 20; old age (O): ≥7 years; n: 8). The sperm-rich portion of the ejaculate from each dog was divided into four aliquots and extended with either tris as a control (C) or tris loaded with 0.5, 1.0, and 1.5 mg/120 × 106 CLC as low (L), intermediate (I), and high (H) doses, respectively. Following equilibration for at least half an hour, the straws were frozen in nitrogen vapor and then stored in liquid nitrogen at least for 48 h. Later, the frozen straws were thawed in a water bath for spermatological evaluation. Significant differences were observed between different age groups in terms of the spermatological parameters (p < 0.05). The evidence suggests that increasing age is associated with poor in-vitro spermatological parameters and CLC was able to protect the acrosome integrity from cryo-damage during the freeze-thawing process. Better semen freezability characteristics were obtained at young ages, considering the overall parameters.

Effects of arginine and trehalose on post-thawed bovine sperm quality.

The present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 °C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 ± 8.21%), CASA motility (12.2 ± 5.69%) and progressive motility (3.52 ± 2.13%), compared with the controls (43 ± 2.73%, 55.4 ± 6.78% and 33.48 ± 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 ± 3.99% and 44.1 ± 2.18%) compared with the control (13 ± 8.15 and 31.7 ± 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.

Vulvar thermal pattern following synchronization of estrus is linked to fertility after timed artificial insemination in goat.

Aim of this study was to study the vulvar thermal pattern variation during the timed artificial insemination protocol in Angora goat and identify the relationship with the successful rate. Does (36 adult healthy females) were synchronized using PGF2α at the day 0, 11 days of progesterone impregnated sponges intra-vaginally, PMSG 48 h before sponges withdraw (day 11) and the intra-cervical inseminations were carried out 48 h later (Timed Artificial Insemination: TAI) with chilled semen. Vulvar (VST) and perivulvar (PST) areas were considered to evaluate the thermal pattern during the protocol at the day 0 and at the TAI using a thermo camera (E60, FLIR System). Differences of temperature (ΔT) between the surfaces were calculated for each time. The does were monitored for pregnancy, delivery time and prolificacy. Pregnant (P) and non-pregnant (NP) does were compared in terms of VST, PST and ΔT using two ways ANOVA considering time and pregnancy as sources of variability. VST was lower than PST in all the monitored does (P < 0.05) (34.79 ± 0.14 vs 36.58 ± 0.14 °C) and without differences between P and NP at day 0 (35 ± 0.18 vs 36.39 ± 0.22 °C). Significant difference (P < 0.05) between P and NP does was recorded at TAI in terms of VST (33.89 ± 0.31 vs 35.40 ± 0.24 °C) and ΔT (-3.16 ± 0.34 vs -1.62 ± 0.26 °C). In conclusion thermal emission by glabrous surfaces in goat may be used to identify the right response induced by hormonal treatments and to optimize the application of assisted reproductive techniques at the field level.