Evaluation of the relationships between oxidative stress, inflammation, hormonal status and sperm parameters in rats: Canonical correlation analysis.

Many endocrine or non-endocrine factors are involved in sperm production. Although reproductive hormones are very important for the initiation and maintenance of spermatogenesis, other factors, such as inflammation and oxidative stress, affect spermatogenesis. The aim of this study is to evaluate the relationships between sperm parameters and hormones, oxidative stress, and inflammation status. We conducted this study on 40 rats. Sperm parameters (motility, abnormal sperm rate, and dead sperm rate), oxidative stress (malondialdehyde, glutathione, glutathione peroxidase, and catalase), inflammation (NF-κß, TNF-α, IL-1ß, IL-6, and IL-10), and hormone parameters (follicle-stimulating hormone, luteinizing hormone, testosterone, melatonin, and corticosterone) were determined. Relationships between mentioned parameters were investigated by canonical correlation analysis. Canonical correlation coefficients for these data sets (sperm-oxidative stress, sperm-inflammation, and sperm-hormone parameters) were found to be strongly significant (rc= 0.875, p<0.001; rc= 0.868, p<0.001; rc= 0.886, p<0.001, respectively). The rate of explanation of oxidative stress, inflammation parameters and hormones by sperm parameters was 61.80 %, 56.10 % and 63.90 %, respectively. Canonical correlation analysis results have revealed that dead sperm rate is mostly related to nuclear factor-kappa beta (NF-κß), catalase, and corticosterone. CCA, which has taken into account the multiple relationships, has revealed that multidimensional evaluation of data sets can provide important and innovative information to researchers for the assessment of relationships between sperm, oxidative stress, inflammation, and hormone parameters.

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.

Determination of expression patterns of miR-26a, and preimplantation factor levels for early pregnancy detection in nulliparous and multiparous cows.

For maximum productivity in a dairy farm, the earliest and the most accurate detection of pregnancy is essential. The aim of this study was to determine the efficacy of expression patterns of miR-26a, and serum Preimplantation Factor (PIF) levels for pregnancy diagnosis during the early pregnancy in nulliparous and multiparous cows. A total of 60 cows (30 nulliparous and 30 multiparous Holstein cows) were enrolled in the study. Blood samples were collected for miR-26a on days 8 and 16 (D8 and D16), and for the PIF on days 10 and 20 (D10 and D20) following insemination (D0). Pregnancies were determined by ultrasonography on the 28th day after insemination. Expression levels of miR-26a determined by qPCR. PIF levels were assessed by using commercial ELISA kits. All data were analyzed by using the MIXED procedure of SPSS. The expression levels of miR-26a were 6.64 folds higher on D16 in pregnant compared to non-pregnant multiparous cows (p < .05). On D8 and D16, miR-26a expression levels were found higher 13 folds in pregnant compared to non-pregnant nulliparous cows (p < .05). Additionally, miR-26a expressions were higher 5.42 folds (p < .05) on D8, 7.19 folds higher (p < .01) on D16 in pregnant nulliparous and multiparous cows, and were 6.30 folds higher (p < .001) on D8 and D16 according to non-pregnant animals. PIF levels were greater in pregnant animals (p < .05). Analyzing miR-26a on D8 might be considered as sufficient in nulliparous cows. Pregnancy detection in multiparous cows can be made on the 16th day with this method. Furthermore, PIF evaluations may be sufficient on D10 in multiparous cows. Besides, PIF levels and miR-26a expression levels might be used safely in field conditions and clinical applications.

Ultrasonographic patterns of different regions of uterus according to pregnancy in cows and heifers.

This study aimed to detect pregnancy within the first 20 days after artificial insemination by evaluating the ultrasonographic patterns of the different regions of uterus in Holstein heifers and cows. Animals were divided into subgroups according to pregnancy on 28th day as pregnant (heifer, n: 15; cow, n: 15) and non-pregnant (heifer, n: 15; cow, n: 15). Images were taken from the ovulation-side cornu uteri (OSC), non-ovulation-side cornu uteri (NOSC), and the corpus uteri (CU) on alternate days from D0 to D20. The images were evaluated by ImageJ software in terms of mean gray value (MGV), homogeneity (HOM), and contrast (CON) and endometrium thickness (ET). The mean MGV and G*T and P*T interactions, the mean CON and G*T and G*P interactions, the mean HOM and G*P interactions, and the mean ET and G*P interactions were statistically significant (p < 0.05). In receiver operating characteristic analyses, D2-D6 for CON and D2, D6, D8, D16, and D20 for HOM of OSC in cows and D8 and D10 MGV and D18 and D20 ET of OSC in heifers had high relationship with positive pregnancy (p < 0.05). The use of echogenicity evaluations and endometrium thickness measurements in reproductive management seems to be suitable for the prediction of pregnancy in cows and heifers.

The thermographic monitoring in early pregnancy detection in Holstein cows and heifers.

This study aimed to conduct a comprehensive investigation to demonstrate early pregnancy detection using thermography in heifers and cows. A total of 60 heifers (n: 30) and cows (n: 30) were divided into two groups as pregnant (n: 15 heifers, n: 15 cows) and non-pregnant (n: 15 heifers, n: 15 cows) according to the day 28 of gestation. Thermographic images were taken from the vulvar and anal regions on alternate days from D0 to D20. Blood samples were collected to determine estrogen and progesterone concentrations. The mean temperature difference between the anal and vulvar regions (ΔT °C) was used in the statistical analyses. Based on the hormonal profiles, no abnormalities were observed for follicular waves or luteal profiles in heifers and cows. The ΔT °C values between heifers and cows and between days were statistically significant (P < 0.001). In thermographic analyses, the differences observed in other main effects and interactions of the group, sampling time, and pregnancy were not statistically significant (P > 0.05). In receiver operating characteristic (ROC) analyses, it was concluded that the ΔT °C value of ≤ 2.9 °C (100% Se - 61.9% Sp) was highly correlated with pregnancy diagnosis in cows on day six after artificial insemination (AI) (P < 0.001). In conclusion, it was determined that the clinical application of thermography can be used for the detection of pregnancy on day six after AI in cows. However, further studies are needed to determine heifers' thermographic characteristics and profiles.

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.

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.

Fertility comparison of frozen bull semen stored in cryogenic deep freezer (-152 °C) and LN2 container.

The present study aimed to use cryogenic deep freezers that could be a feasible alternative for cryopreserved semen storage. A total of 284 straws from three Simmental bulls and 272 Simmental cows were used. The experimental group consisted of 151 semen straws that were stored at -152 °C for a week. Moreover, the control group consisted of 133 semen straws that were stored at -196 °C. Firstly, two samples per bull (n = 6) were examined in terms of sperm kinetic parameters by CASA. Furthermore, plasma membrane, acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were analyzed by flow cytometry. Then, artificial inseminations were performed on Simmental cows with 272 straws belonging to two groups. Then, 56th-day Non-return Rate (NRR56) was determined. All spermatological data were subjected to a linear mixed model. Chi-Square test was performed to NRR56 between storage temperature groups. Also, logistic regression analysis was used to examine the effect of bull, storage temperature and age of cows on pregnancy status. While age of cows was included in the final logistic regression model, effect of bull x storage temperature was not included because it was found as non-significant. The post-thaw PMOT and STR of cryopreserved bull semen, which was stored at -152 °C, had lower and statistically significant values (p < 0.05). However, frozen bull semen, which were stored at -152 °C, kept its fertility ability as which stored at -196 °C. Besides, NRR56 of semen stored at -152 °C and -196 °C were detected as 57.24% (83/145) and 55.91% (71/127), respectively (p > 0.05). Nevertheless, these results should be supplemented with more pre-freezing and post-thaw sperm quality analyses and more fertility data for increasing the accuracy of the method.

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.

Effects of Spermine and Spermidine supplemented extenders on post-thaw Spermatological Parameters in Stallion Semen Cryopreservation.

In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared with different doses of Spermine and Spermidine (0,1 mg/ml; 0,2 mg/ml; 0,4 mg/ml; 1 mg/ml; 2 mg/ml; 4 mg/ml). Stallion semen that were cryopreserved with Control and experimental extenders were evaluated in terms of Total Motility, Progressive Motility, Plasma Membrane Integrity, Capacitation Index, Acrosome Integrity and DNA Fragmentation Index. At the end of the evaluations, it was determined that 0,2 mg/ml Spermine and 0,4 mg/ml Spermidine showed better Total Motility and Progressive Motility, numerically. On the other hand, it was observed that 4 mg/ml Spermine and Spermidine had the lowest statistically significant values (p < 0,001). While statistically similar differences were obtained between groups in terms of the Plasma Membrane and Acrosome Integrity, it was determined that all experimental groups had lower and statistically significant values in terms of Capacitation and DNA Fragmentation Index (p < 0,001). As result, it was observed that the stallion semen cryopreservation success can be increased by the addition of 1-2 mg/ml Spermine that had effective protection on Capacitation and DNA Fragmentation Index without damaging other spermatological properties.

Semen collection by trans-rectal digital stimulation and insemination campaign in goat.

The overall purpose of this study was to describe a method of semen collection via trans-rectal digital massage (TDM) and to carry out a related fertility trial in Angora goat. Sixteen Angora bucks (ranging 1-4 years) and 28 nulliparous does (1-2 years) were used in this study. Semen samples were collected via trans-rectal massage from 85.71% of the bucks in multiple attempts (18/21). The mean values of volume, pH, mass motility, total motility, concentration, viability, abnormal spermatozoa rate and ejaculation time were 0.64 ± 0.09 ml, 6.3 ± 0.21, 2.7 ± 0.34, 58.18 ± 5.1%, 3.68 ± 0.31 × 109 /ml, 71.38 ± 7.12%, 18.22 ± 2.48% and 3.4 ± 0.33 min respectively. Oestrus was detected with teaser buck and confirmed by using infrared thermography and ultrasonography (US). The success rate of synchronisation was found as 71.4% (20/28). On Day 21, pregnancy diagnosis was performed trans-rectally with US and the pregnancy rate was determined as 78.57% (11/14). TDM method of semen collection seems to be easily applicable to the buck and it could be a good alternative to collect semen as well as its use in artificial insemination campaign. Thermal monitoring is found to be a valuable tool to monitor the response to hormonal driven ovulatory synchronisation in Angora does during timed artificial insemination.

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.

The use of infrared thermography to detect the stages of estrus cycle and ovulation time in anatolian shepherd dogs.

BACKGROUND: The aim of the study is to evaluate the effectiveness of thermographic monitoring, using the temperature changes of perianal and perivulvar areas for the determination of estrus in Anatolian Shepherd bitches. Fifteen bitches were used in the study. Blood and vaginal smear samples were collected and thermographic monitoring of perianal and perivulvar areas were carried out starting from proestrus to early diestrus. Also, external signs of estrus were investigated. Smear samples were evaluated by light microscopy after Diff-Quik staining method and superficial and keratinized superficial cells were determined as percentage (S + KS%). Progesterone and luteinizing hormone measurements were done by radioimmunoassay. The difference in temperature between perianal and perivulvar areas was evaluated through thermographic images by FLIR ResearchIR Software. RESULTS: According to the results obtained from the study, differences between progesterone and S + KS% were statistically significant (P < 0,05). Although temperature showed increase and decrease with progesterone and S + KS%, the differences were not important statistically (P > 0,05). Serum luteinizing hormone levels did not sign any difference (P > 0,05). CONCLUSIONS: As a result, thermographic monitoring alone is not enough for estrus detection in Anatolian Shepherd bitches. However, it can be used to assist the actual estrus detection technique in terms of providing some foreknowledge by evaluating the differences in temperature.

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.