Abbas Farshad

Sperm cryopreservation is widely used in reproductive biotechnology, but the freeze–thaw process often reduces sperm quality by impairing motility, viability, and DNA integrity. This study assessed how antifreeze protein III (AFP III; 1.5 µg/mL) and trehalose (25 mM), alone or combined, influence post-thaw sperm quality in swim-up fractions. After routine semen evaluation, ejaculates were separated into upper, native, and lower fractions and diluted in a Tris–fructose–egg yolk extender containing the additives. Post-thaw analyses covered motility, kinematic parameters, apoptosis, intracellular hydrogen peroxide (H₂O₂), mitochondrial activity, lipid peroxidation, viability, and DNA fragmentation. Because all fractions originated from the same ejaculate, differences among native, upper, and lower sperm populations were evaluated using a mixed-effects model with sperm fraction as a fixed effect and dog/ejaculate as a random effect. Native semen showed significant differences among fractions (p < 0.05). The upper fraction showed the highest TM and PM, together with clearly superior kinematic performance in VCL, VSL, VAP, and ALH. All these parameters were significantly higher than those measured in the lower fraction (p < 0.05). In contrast, the native fraction displayed intermediate values for TM, PM, VCL, VSL, VAP, and ALH, showing a significant difference compared with the upper fraction (p < 0.05) but no significant difference relative to the lower fraction (p > 0.05). Beat cross frequency (BCF) did not differ among groups (p > 0.05). AFP III improved motility and reduced H₂O₂-derived ROS (p < 0.05), whereas trehalose decreased motility (p < 0.05) but increased mitochondrial activity in the lower fraction (p < 0.05). After freezing, the upper fraction showed higher lipid peroxidation and DNA fragmentation, while the lower fraction exhibited more apoptosis and reduced viability (p < 0.05). Overall, AFP III provided the most consistent protection, and trehalose produced mixed, fraction-dependent effects, confirming that sperm subpopulation characteristics strongly influence freeze–thaw outcomes.

Background: Accurate assessment of sperm morphology is essential for evaluating the quality of cryopreserved canine semen used in artificial insemination and for improving cryopreservation protocols. This study compared six staining techniques, Eosin, Eosin–Nigrosin, Diff-Quick^®, Hemacolor^®, Spermac^®, and Formol-citrate Bengal Rose, for light-microscopic evaluation of frozen–thawed canine spermatozoa. Methods: Semen from ten dogs was thawed, divided into four aliquots, and either left untreated or exposed to thermal stress at 6 °C, 18 °C, or 37 °C for two hours to induce morphological variation. A total of 360 slides and 960 evaluations were performed immediately after staining and again after 24 h, 7 days, and 3 months to assess staining quality and stability over time. Results: Eosin produced stable staining for up to three months and was the most economical method, though its initial detail recognition was lower than that of Spermac^® and Formol-citrate Bengal Rose. Eosin–Nigrosin showed reduced contrast and detail. Diff-Quick^® provided better contrast than Eosin–Nigrosin, while Hemacolor^® maintained consistent quality regardless of stress treatment or storage duration. Spermac^® yielded the highest initial morphological detail but deteriorated during storage. Formol-citrate Bengal Rose combined high detail recognition with stable staining throughout the study. Cryopreservation increased looped tails, and incubation at 37 °C markedly elevated pathological sperm and rudimentary tails. Conclusions: All six staining methods were suitable for evaluating and archiving frozen–thawed canine semen. Formol-citrate Bengal Rose and Spermac^® offered the best detail, while Eosin provided a cost-effective option with excellent long-term stability.

Background and Aim: Dairy goats exhibit distinct physiological adaptations during pregnancy and lactation, necessitating breed-specific biochemical reference values for accurate health assessment. The Thuringian Forest goat, an important regional dairy breed managed under organic systems, remains insufficiently characterized in this regard. This longitudinal study aimed to evaluate clinical-chemical profiles and metabolic dynamics across reproductive stages and to determine the influence of lactation number, milk yield, and litter size on biochemical parameters. Materials and Methods: A total of 25 clinically healthy Thuringian Forest dairy goats were monitored over one production cycle under standardized organic farming conditions. Blood samples were collected monthly from late gestation (−3 to −1 months) through 12 months of lactation, yielding 300 serum samples. Analyses included enzyme activities (alkaline phosphatase, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, and creatine kinase), electrolytes (sodium, potassium, calcium, phosphate, and magnesium), and metabolic indicators (glucose, cholesterol, β-hydroxybutyrate, urea, creatinine, total protein, triglycerides, and free fatty acids). Repeated measures analysis of covariance was used to assess the effects of time, lactation number, milk yield, and litter size. Results: Marked physiological variations were observed across reproductive stages. Sodium and potassium decreased slightly prepartum and fluctuated significantly during lactation, while calcium, phosphate, and magnesium showed stage-dependent changes. Enzyme activities exhibited clear lactation-related patterns, with alkaline phosphatase increasing progressively, gamma-glutamyl transferase peaking in early lactation, and aspartate aminotransferase declining over time. Metabolic parameters demonstrated adaptive responses, including increased glucose, total protein, and cholesterol during lactation, and elevated β-hydroxybutyrate in early lactation without exceeding clinical thresholds. Milk yield significantly influenced several biochemical markers, including sodium, creatine kinase, triglycerides, and β-hydroxybutyrate, whereas litter size had minor effects, primarily on sodium and urea concentrations. No evidence of pathological alterations was detected. Conclusion: The study provides comprehensive longitudinal data on biochemical and metabolic adaptations in Thuringian Forest dairy goats under organic management. Lactation stage and milk yield are the primary determinants of biochemical variation, while litter size plays a limited role. These findings support the development of breed-specific reference intervals and highlight the importance of considering physiological and production factors for accurate clinical interpretation and improved herd management. Keywords: biochemical profile, dairy goats, lactation, metabolic adaptation, milk yield, organic farming, pregnancy, Thuringian Forest goat.

Background and Aim: Cryopreservation is commonly used in canine reproductive biotechnology to preserve genetic material for long-term storage. However, the freeze–thaw process induces oxidative stress, mitochondrial dysfunction, and structural damage in spermatozoa, thereby reducing post-thaw sperm quality and fertility potential. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, has been suggested as a promising cryoprotective additive that mitigates mitochondrial reactive oxygen species and enhances cellular integrity. This study aimed to assess the dose-dependent effects of MitoQ supplementation on mitochondrial function, oxidative stress, and the post-thaw quality of cryopreserved canine spermatozoa to identify an optimal concentration for semen cryopreservation. Materials and Methods: Semen samples were collected from 10 healthy and fertile male dogs of various breeds. Each ejaculate was divided into four equal parts and cryopreserved with a Tris–fructose–egg yolk extender supplemented with different MitoQ concentrations (0, 100, 200, or 400 nM). After four weeks of storage in liquid nitrogen, the samples were thawed and evaluated in vitro. Post-thaw sperm quality was measured using computer-assisted sperm analysis to assess motility and kinematic parameters. Additional evaluations included sperm viability, plasma membrane integrity, acrosome integrity, lipid peroxidation levels, apoptotic status via flow cytometry, mitochondrial membrane potential, and intracellular hydrogen peroxide production. Data were analyzed using a mixed-effects model with dog as a random effect, and significance was set at p < 0.05. Results: MitoQ supplementation had concentration-dependent effects on several sperm quality parameters. The 200 nM MitoQ group showed the most consistent improvements in post-thaw sperm quality. Total motility and curvilinear velocity were significantly higher at this concentration compared to the control and other treatment groups. Additionally, sperm treated with 200 nM MitoQ displayed improved plasma membrane and acrosome integrity, reduced lipid peroxidation, and a lower proportion of dead sperm cells. Trends toward increased viability and enhanced mitochondrial activity were also seen at this concentration. In contrast, 400 nM MitoQ supplementation was linked to decreased membrane integrity and increased oxidative stress markers, suggesting possible pro-oxidant effects at higher doses. Overall, the results indicate that moderate MitoQ supplementation supports mitochondrial redox balance and partially reduces cryo-induced damage in canine spermatozoa. Conclusion: MitoQ supplementation at 200 nM modestly but consistently enhanced several in vitro indicators of post-thaw sperm quality in dogs, such as motility, membrane stability, and oxidative stress markers. These findings highlight the narrow therapeutic window of mitochondria-targeted antioxidants and stress the importance of dose optimization during semen cryopreservation. Although the observed improvements suggest potential benefits for reproductive biotechnology, further research with fertility-related functional outcomes and larger sample sizes is needed to confirm the practical reproductive impact of MitoQ supplementation in canine semen cryopreservation. Keywords: canine sperm cryopreservation, mitoquinone, mitochondrial function, oxidative stress, post-thaw sperm quality, reactive oxygen species, sperm motility, sperm viability.

Background and Aim: Pregnancy and lactation place significant physiological demands on dairy goats, affecting red blood cell (RBC) indices, white blood cell (WBC) profiles, and platelet traits. Despite the diagnostic value of hematology (HA), there are no longitudinal, breed-specific reference values for Thuringian Forest goats. This study aimed to describe changes over time in differential blood counts and platelet indices in clinically healthy does kept under BIOLAND-certified organic management. Materials and Methods: A longitudinal study was conducted over one year using 25 clinically healthy Thuringian Forest does. Monthly blood samples were collected from 3 months prepartum through 12 months postpartum, resulting in 295 samples. Hematological analyses included RBC count, hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular HGB (MCH), mean corpuscular HGB concentration (MCHC), RBC distribution width (RDW), and HGB distribution width (HDW). Platelet parameters, platelet count (PLT), mean platelet volume (MPV), and platelet distribution width (PDW), and differential WBC counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) were measured using a validated automated analyzer. Repeated-measures analysis of variance evaluated the effects of reproductive stage, parity, milk yield, milk composition, and litter size. Results: Significant stage-dependent hematological changes were observed. RBC, HGB, and HCT decreased during late gestation and reached their lowest levels before birth, then increased gradually during lactation. MCV and MCH remained stable. PLT increased around parturition, MPV declined before birth and rose after, and PDW decreased steadily from late gestation through lactation. Neutrophils and total WBC counts increased toward parturition, while lymphocytes and monocytes rose during lactation. Eosinophils peaked at the start of lactation, and basophils declined after birth. Parity and milk yield significantly influenced certain RBC, platelet, and leukocyte parameters, whereas litter size showed no significant effect. Conclusion: Thuringian Forest goats show unique hematological changes during pregnancy and lactation, reflecting metabolic, hormonal, and immune adjustments related to reproduction and milk production. These breed-specific, stage-specific reference values improve clinical interpretation and diagnosis in dairy goat management. To our knowledge, this is the first longitudinal hematological study of this breed in organic farming conditions. Keywords: blood parameters, dairy goats, hematology, lactation, organic farming, pregnancy, Thuringian Forest breed, veterinary diagnostics.

Sperm cryopreservation is a key technique in assisted reproductive technologies (ART), livestock breeding, fertility preservation, and wildlife conservation. However, the freeze–thaw process induces significant oxidative stress through the production of reactive oxygen species (ROS) by mitochondria, which can lead to impaired sperm motility, membrane damage, DNA fragmentation, and reduced fertilization potential. MitoQ is a mitochondria-targeted antioxidant consisting of a ubiquinone moiety conjugated to triphenylphosphonium (TPP⁺). MitoQ selectively accumulates in the mitochondrial matrix, where it efficiently scavenges reactive oxygen species (ROS) at their point of origin. This targeted action helps preserve mitochondrial function, sustain ATP production, and inhibit apoptotic signaling. Extensive experimental evidence across diverse species, including bulls, rams, boars, humans, dogs, and goats, shows that MitoQ supplementation during cryopreservation enhances post-thaw sperm viability, motility, membrane integrity, and DNA stability. Optimal dosing between 50 and 150 nM achieves these benefits without cytotoxicity, although higher doses may paradoxically increase oxidative damage. Compared to conventional antioxidants, MitoQ offers superior mitochondrial protection and enhanced preservation of sperm bioenergetics. Future directions involve exploring synergistic combinations with other cryoprotectants, advanced delivery systems such as nanoparticles and hydrogels, and detailed mechanistic studies on long-term effects. Overall, MitoQ represents a promising adjunct for improving sperm cryopreservation outcomes across clinical, agricultural, and conservation settings.

Accurate assessment of sperm morphology is essential for artificial insemination using liquid-preserved boar semen. This study compared nine commonly used staining techniques, eosin, eosin–nigrosin, Diff-Quick^®, Hemacolor^®, Sangodiff-G^®, Spermac^®, Formol–Citrate–Rose Bengal stain, Testsimplets^®, and Methyl Violet, based on morphological assessment, cost, time efficiency, and storage stability. Each staining method was applied to 36 slides, totaling 324 samples, and evaluated four times each (1296 evaluations). Slides were analyzed four times: immediately after staining and after 1 day, 1 week, and 3 months of storage. The results indicated that Eosin was the fastest and most cost-effective method, providing strong contrast, though it increased detection of structural alterations. Eosin–nigrosin offered detailed morphology but formed colored crystals over time. Diff-Quick^® and Hemacolor^® showed good initial performance, but Hemacolor^® lost pigment clarity after 3 months (p = 0.0273). Sangodiff-G^® had poor contrast and reduced detection of abnormalities (p = 0.00229). Spermac^® delivered high contrast but was time-consuming. Formol–Citrate–Rose Bengal stain required extensive preparation and showed significant post-storage changes (p < 0.0001). Testsimplets^®, despite their high cost, suffered from declining interpretability (p < 0.0001). Methyl Violet lacked sufficient resolution and was highly unstable over time (p < 0.0001). In conclusion, Eosin emerged as the most practical and economical staining method for routine morphological evaluation of liquid-preserved boar semen. While eosin–nigrosin was also effective, its storage instability limits broader application. Other methods showed specific weaknesses, emphasizing the need to tailor stain selection to laboratory goals and constraints.

(1) Background: Cryopreservation of epididymal spermatozoa in dogs is challenging due to their lower cryotolerance compared to ejaculated spermatozoa. Given the limited sperm volume obtained per individual, efficient recovery and preservation techniques are essential. (2) Methods: This study assessed sperm collection and cryopreservation methods from the cauda epididymis of ten dogs undergoing routine elective castration. After dissection and mincing, the cauda epididymidis tissue was incubated in 0.9% saline at 38 °C for either 10- or 30-min. Samples were analyzed for concentration and motility using AndroVision^® software (CASA; AndroVision™; Minitüb GmbH) (Tiefenbach, Germany). Additional evaluations included histological examination, hypoosmotic swelling test, live/dead staining, and morphological assessments. Three extenders, custom-made Tris-Fructose-Citrate (Tris), custom-made Uppsala, and commercial Optixcell^®, were used for cryopreservation and compared for post-thaw sperm quality. (3) Results: No significant differences were found between the 10- and 30-min incubation groups regarding sperm motility, viability, or histological integrity. The total sperm counts were 292 × 10⁶ ± 175 × 10⁶ for the 10 min group and 233 × 10⁶ ± 162 × 10⁶ for the 30 min group (p = 0.56). Histological sections revealed no significant difference in residual intraluminal spermatozoa between groups, indicating that 10 min of incubation is sufficient for effective sperm migration. Post-thaw sperm motility was significantly higher with Uppsala (17.2 ± 12.2%) and Optixcell^® (11.7 ± 6.5%) compared to Tris (4.7 ± 4.8%). Morphological abnormalities were lowest in samples preserved with Optixcell^® (37.5 ± 10.1%, p = 0.005). (4) Conclusion: A 10 min incubation period is adequate for efficient recovery of epididymal sperm in dogs. Among the tested extenders, Uppsala and Optixcell^® demonstrated superior cryoprotective effects, resulting in better post-thaw motility and reduced morphological abnormalities compared to Tris.

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.