κ-Carrageenan is a plant polysaccharide derived from red seaweeds reported to possess potential medicinal and antioxidants activities. The present study aimed to identify the cryoprotective effects of κ-carrageenan on the quality of frozen-thawed canine semen. Twenty-eight ejaculates were collected and diluted in a Tris egg-yolk-free extender supplemented with various concentrations of κ-carrageenan (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). The addition of κ-carrageenan to the extender at a 0.2% concentration induced a significant increase in the total motility (TM) and the rapid progressive motility (RPM) of canine sperm. Among the experimental groups, the highest percentage of sperms with intact acrosomes was found in the 0.5% κ-carrageenan group (p < 0.05). Apoptosis levels were significantly lower in the 0.1% and 0.2% κ-carrageenan treatment. Moreover, sperm in the κ-carrageenan supplemented group showed a significantly higher expression of antiapoptotic (Bcl-2) and lower expression of NADPH oxidase (NOX5), spermine synthase (SMS), and spermine oxidase (SMOX) genes than those in the control group. In conclusion, the addition of κ-carrageenan to the freezing extender improved the overall efficiency of frozen-thawed dog spermatozoa.

The use of chilled semen has gained increasing interest in canine reproductive services. The addition of phosphodiesterase (PDE) inhibitors that increase the intracellular cyclic adenosine monophosphate levels may improve sperm motility. The purpose of this study was to examine the quality of sperm under the effect of the specific PDE-10 inhibitor (papaverine) added after storage for 1, 2, and 3 days at 5 °C. The ejaculates were obtained from 5 healthy Beagle dogs by digital manipulation. After collection, ejaculates were pooled, extended and cooled at 5 °C during 3 days. Sperm parameters were tested 30 min after the addition of different papaverine (PA) concentrations: 0, 5, 10 and 20 µM. Sperm motility (CASA), viability (PI/FITC-PNA) and capacitation status (chlortetracycline assay) were evaluated. The results showed that the addition of PA has no effect on sperm samples at day 0. However, concentrations of 5 and 10 µM increased (p < .05) sperm motility kinetics and viability significantly compared to the control at day 1, day 2 and day 3 of cooling. The addition of 20 μM PA decreased (p < .05) sperm quality parameters significantly and increased the percentage of capacitated/acrosome-reacted spermatozoa. In conclusion, the addition of 5 and 10 μM PA concentrations after cooled storage improved canine sperm quality.

Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r2 = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

Sperm cryopreservation is an assisted reproductive technique routinely used in canine species for genetic conservation. However, during cryopreservation, the DNA damages are still elevated, limiting the fertilization rate. The present study was conducted to evaluate whether supplementation of canine semen extender with a molecule limiting the metabolic activities can improve the quality of frozen-thawed canine spermatozoa. We used metformin, known to limit the mitochondrial respiratory and limit the oxidative stress. Before and during the freezing procedure, metformin (50µM and 500µM) has been added to the extender. After thawing, sperm exposed to metformin conserved the same viability without alteration in the membrane integrity or acrosome reaction. Interestingly, 50µM metformin improved the sperm motility in comparison to the control, subsequently increasing mitochondrial activity and NAD+ content. In addition, the oxidative stress level was reduced in sperm treated with metformin improving the sperm quality as measured by a different molecular marker. In conclusion, we have shown that metformin is able to improve the quality of frozen-thawed dog semen when it is used during the cryopreservative procedure.

Introduction The addition of low-molecular-weight antioxidants during the freezing process improves post-thaw sperm quality. The high antioxidant potential of cryopreserved semen could have a positive effect on the motility, viability, and energy status of sperm cells and their ability to bind to the zona pellucida of oocytes. The aim of the study was to determine the effects of different concentrations and combinations of vitamins E and C in a semen extender on selected quality parameters of frozen-thawed canine spermatozoa. Material and Methods The experimental material was the semen of four mixed-breed dogs. Sperm viability (motility, plasma membrane integrity, and mitochondrial function) was examined at 0, 60, and 120 min in semen samples supplemented with the extender and in the controls. Results Combined supplementation with vitamins C + E at a concentration of 200 + 200 μM /1 × 109 spermatozoa had the most profound effect on total sperm motility, linear motility, and the percentage of spermatozoa with intact plasma membrane and active mitochondria. Conclusion The synergistic activity of vitamins E and C had a more beneficial influence on the quality of frozen-thawed sperm than these non-enzymatic antioxidants applied separately.

This study investigated the correlation between oxidative stress status and key canine sperm parameters and the effect of addition of a superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) combination in egg yolk tris-citrate glucose (EYT-G) extender on semen during 10 days of storage at 4℃. Ten Boxer dogs were divided into two groups, fertile (F) and hypofertile (H), depending on pregnancy and live birth rate status in the previous year. Semen evaluation was performed on the day of collection (D0) and after 5 (D5) and 10 (D10) days of cooled storage. Sperm motility, kinetic parameters, and DNA integrity were assessed. A correlation between oxidative status and key semen parameters in both F and H groups was observed. Total and progressive motilities were significantly higher in the treated (SOD, CAT, and GPx addition) versus control groups at D10 in both F and H groups, and at D5 in the H group. DNA integrity was significantly higher in both treated groups (H and F) at D5 and D10. In conclusion, the addition of SOD, CAT, and GPx in the extender allows preservation of semen quality for up to 10 days of storage at 4℃ in both fertile and hypofertile dogs.

Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.

Semen quality in dogs has not been assessed in a longitudinal study that includes endpoints of female fertility and pregnancy. Although use of artificial insemination with chilled semen is increasingly used in canine reproduction, the resultant level of predictability and odds of fertile matings for dogs is still not fully understood. This research provides, for the first time, comprehensive semen evaluation in a large population of dogs in which fertility has been tracked. Duplicate ejaculates were obtained from 39 Labrador retriever males of the Guide Dogs for the Blind (San Rafael, CA, USA) breeding program. Sperm endpoints were determined in fresh semen and extended chilled semen at 48 hour after collection. Evaluation included total and progressive motility, average path velocity, morphology, membrane lipid peroxidation, presence of sperm reactive oxygen species, sperm chromatin structure, and mitochondrial DNA copy number. Male age ranged from 1 to 10 years and were grouped as young (Y; 1-3 years, n = 21), middle aged (M; 4-6 years, n = 13), and senior (S; 7 years or greater, n = 5) for analysis. The effects of age and sperm state (fresh vs. chilled) on the above sperm endpoints were determined using a linear mixed effects model. Semen endpoint values for all parameters were established for this group of fertile males. Progressive motility was only lower in the senior male chilled samples compared to all other groups, fresh and chilled (P < 0.05). Velocity decreased with increasing age and was lower overall in chilled samples (P < 0.05). Percent morphologically normal sperm was lower in senior dogs compared with the other age groups (P < 0.05). The presence of reactive oxygen species was lower in chilled samples compared with fresh (P < 0.05). For sperm chromatin structure, the senior-aged group had a higher %COMPαt than the middle-aged group (P < 0.05). Bayesian analysis determined that no differences were seen in total motility, membrane lipid peroxidation, and mitochondrial DNA copy number, with regard to conception rate or average litter size between age groups or between fresh and chilled samples. We observed no effects from semen quality on fertility or fecundity regardless of age, despite the differences found in semen quality. The use of advanced laboratory tests to evaluate sperm parameters beyond the standard motility, morphology, and concentration will open investigation to more specific and sensitive fertility tests in canine reproduction.

This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4°C) canine sperm over a period of 4 days. In the first experiment, 20 ejaculates collected from 5 Beagle dogs were chilled in an extender containing 6% low density lipoproteins (LDL) (Control), or one of 7 extenders containing different concentrations (2, 4, 6, 8, 10, 15, 20%) of liposomes (LIPO). These ejaculates were chilled over 4 days and motility was assessed daily using a Hamilton Thorne analyzer (HTM-IVOS, 14.0). The 2% LIPO obtained the best results (p=0.038) after four days (72.55% motile spermatozoa and 31.4% progressive spermatozoa). In experiment 2, 10 ejaculates were collected from same 5 dogs and chilled in 6% LDL or 2% LIPO-based extenders. Sperm integrity characteristics were assessed prior to refrigeration and every 48h for four days (D0, D2, and D4). Acrosome integrity was assessed using the FITC-PSA test (Fluorescein IsoThiocyanate-Pisum Sativum Agglutinin), plasma membrane (PM) integrity using both the hypo-osmotic swelling test (HOSt) and SYBR14/Propidium Iodide test (SYBR14/PI), and DNA integrity using the Acridine-Orange test (AO). The 2% LIPO extender provided equivalent preservation of sperm integrity parameters to the reference extender (6% LDL). In experiment 3, a Langmuir-Blodgett trough was used to evaluate the mechanistic interactions between LDL, LIPO, prostatic fluid, and the canine spermatozoal membrane during chilling. Results indicate that LDL and LIPO interact differently with the biomimetic membrane. The most likely conclusion of these findings is that LDL and liposomes employ different protective mechanisms during the chilling (4°C) of canine spermatozoa.