Methyl ß-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production.

BACKGROUND: Generating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl ß-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm. RESULTS: In this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique. CONCLUSION: Overall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals.

Effects of short-term oral letrozole on fresh semen parameters, endocrine balance, and prostate gland dimensions in domestic dogs.

BACKGROUND: Aromatase inhibitors improve male fertility by modifying the hormonal control of spermatogenesis. The present study aimed to investigate the effects of oral administration of letrozole on testosterone and estradiol concentrations and their ratios in blood serum, seminal plasma, prostatic fluid, sperm quality in fresh semen, and prostate gland dimensions. Seven adult male intact mixed-breed dogs were selected. The animals received letrozole (72 µg/kg, PO) daily for four weeks. Blood samplings and semen collections were carried out on days 0 (control), 14 (treatment), 28 (treatment), and 42 (post-treatment). RESULTS: Our results showed that letrozole administration resulted in a 4.3 fold significant increase in serum, seminal plasma, and prostatic fluid testosterone levels after 14 days. This remained high until the end of the study. Serum and prostatic fluid estradiol levels did not change significantly over the study period. However, the seminal plasma estradiol level showed a significant increase on day 14. The estradiol: testosterone ratio was significantly reduced on day 14 in serum, seminal plasma, and prostatic fluid samples. Letrozole significantly improved the ejaculated spermatozoa viability and concentration after 28 days of oral administration. However, the sperm plasma membrane functional integrity and kinematic parameters were not significantly affected by the treatment. Transabdominal ultrasound examination revealed a significant increase in the height, width, and volume of the prostate gland after 28 days of treatment. CONCLUSIONS: According to the present research, oral administration of letrozole for 28 days affects local and systemic sex hormone balance leading to an improvement of the ejaculated canine spermatozoa viability and concentration concurrent with an increase in the prostate gland dimensions.

Effect of direct therapeutic ultrasound exposure of ovaries on histopathology, inflammatory response, and oxidative stress in dogs.

BACKGROUND: This research was designed to evaluate the effects of therapeutic ultrasound waves on ovarian germinal tissue and inflammatory cytokines (interleukin-6 (IL-6), IL1ß, tumor necrosis factor-α (TNF-α)), acute phase proteins (serum amyloid A (SAA), C reactive protein (CRP)) and oxidative stress (total antioxidant capacity (TAC), and malondialdehyde (MDA)) in dogs. Twenty-six clinically healthy adult mix-breed female dogs were aligned into three groups. Laparotomy was performed in control (n = 6) and treatment (T5, n = 10; T10, n = 10) groups. The ultrasonic exposure of ovaries in treatment groups was performed during laparotomy by round motions of the therapeutic ultrasonic transducer on both ovaries (1 MHz frequency, 1.5 W/cm2) for 5 min in the T5 group and for 10 min in the T10 group. Blood samples were collected from the jugular vein into a plain glass tube on days 0 (before laparotomy), 3, 6, and 9 after surgery. All control and treatment groups' dogs were ovariectomized for histological evaluation on day 60 after laparotomy or laparotomy + ultrasound exposure. RESULTS: Direct exposure of ovaries with therapeutic ultrasound waves induced inflammation and oxidative stress comparison with the control group. Histopathological evaluation of treated ovaries with ultrasound waves indicated a decreased number of primordial follicles (ovarian reserve) and oocyte preservation scores compared with ovaries in the control group. CONCLUSIONS: These changes may cause subfertility in the long term. It seems that inflammatory response and oxidative stress are factors in the permanent damage of ovarian tissue.

Effects of extender filtration and egg yolk concentration on canine semen cryopreservation.

The semen cooling and freezing extenders commonly contain the chicken egg yolk (EY) as the main sperm cryoprotectant. Besides its advantages, the EY has large lipoprotein granules that cause several physical and biological interferences. The previous studies have proposed several methods to resolve the problems with the EY-based semen extenders, including mechanical agitation, EY fractionation, replacing the EY with purified EY LDL, and ultrasonication. In the current research, we aimed to evaluate the syringe filtration (220 nm) of an EY-based canine semen freezing extender as a simple and cheap method to remove the EY granules. We also studied the possibility of re-aggregation of EY granules after cooling, freeze/thawing, and lyophilization/rehydration of the filtered extenders. Additionally, we compared the effects of the filtration on lipid profile, turbidity, EY particle size distribution, and osmolality of the EY-based extenders. Next, we examined the effects of filtered extenders containing different levels of EY (5%, 10%, 15%, 20%, and 25%) versus the control extender (20% EY, unfiltered) on post-thaw sperm quality traits. We collected the semen samples from seven clinically healthy mixed-breed adult dogs and pooled them for sperm freezing procedures. Samplings were repeated at least five times, independently. Our results indicated that the syringe filtration could remove the large EY particles and reduce the extender turbidity without affecting the lipid profile of the whole extender solution. The filtered extender supplemented with 25% (v/v) EY led to the best post-thaw canine spermatozoa quality markers. The frozen-thawed spermatozoa evaluations included motility parameters (computer-assisted semen analysis system), membrane and acrosome integrity (hypo-osmotic swelling test, chlortetracycline binding assay), DNA fragmentation (sperm chromatin dispersion assay), membrane lipid peroxidation (MDA levels), apoptosis (Annexin V/propidium iodide assay), and fertility-associated sperm mRNA transcript abundance (protamine 2 and 3). In conclusion, the syringe filtration of the EY-based semen extenders was a simple and cheap method that could effectively remove large EY lipoprotein granules and possibly prevent EY-origin bacterial contamination of the final extender solution. The EY at 25% (v/v) concentration in the filtered extenders resulted in the highest canine spermatozoa cryo-tolerance.

Supplementation of melatonin to cooling and freezing extenders improves canine spermatozoa quality measures.

BACKGROUND: Sperm freezing and cold storage are the two most common assisted reproductive technologies in the canine breeding industry. The freeze-thawing process causes significant detrimental changes in both sperm cell structure and function. Previous research has confirmed that excessive accumulation of un-scavenged free radicals (oxidative stress) plays an important role in the cryopreservation-induced damage to sperm cells. Also, the gradual accumulation of the free radicals during cold storage leads to a decline in the sperm quality markers. Melatonin is an endogenous neurohormone synthesized from tryptophan amino acid by pineal glands. Besides its several well-known physiologic roles, melatonin has a significant antioxidant potential through direct free radical scavenging properties. Therefore, the current study was designed to evaluate the potential in vitro protective properties of melatonin (0.5, 1, and 2 mM) on canine sperm cells after freezing or during long-term cold storage (9 days, 5 °C) on most important sperm in vitro fertility markers. RESULTS: Melatonin at 0.5, 1- or 2-mM concentrations could preserve significantly higher sperm total motility after 4 days of cold storage. However, only the 1- and 2 mM melatonin concentrations could result in better TM and PM values after 7 days of cold storage. Furthermore, melatonin supplementation could preserve higher sperm viability and acrosome integrity after 7 days of storage. Also, it could have significant protective effects on the cooled sperm DNA integrity. In the freezing section of the current research, melatonin at either 1- or 2-mM concentrations could not improve the sperm post-thaw TM and PM, whereas they improved sperm DNA integrity. Also, the post-thaw plasma membrane functional integrity and sperm velocity parameters were not affected by the treatment. Although DMSO (Dimethyl Sulfoxide) as the melatonin solvent could reduce the level of sperm lipid peroxidation and even improve the post-thaw sperm DNA integrity compared to the negative control, it reduced the post-thaw sperm progressive motility. However, the negative effects were reversed by concurrent melatonin supplementation at 1- and 2-mM concentrations. CONCLUSION: The addition of 1- or 2-mM melatonin to the canine sperm freezing and cooling media could improve sperm motility, viability, acrosome, and DNA integrity.

Effects of short-term administration of melatonin before gonadectomy on oxidative stress, cortisol and sex hormones in male dogs.

The aim of this study was to investigate gonadectomy stress, steroid hormones and serotonin in male dogs treated with melatonin before gonadectomy. Twenty-five mixed breed adult dogs were divided into five equal groups. The melatonin and melatonin + gonadectomized groups received melatonin treatment (3 mg/10 Kg, PO, TID) the day before gonadectomy; the gonadectomized and anaesthesia groups did not receive melatonin; and the control group just received the melatonin vehicle. Blood sampling was performed before melatonin administration (day -1) and on days 0 (gonadectomy), 1, 3 and 6 after gonadectomy. Superoxide dismutase and glutathione peroxidase concentrations decreased significantly in gonadectomized dogs compared with dogs treated with melatonin before gonadectomy and intact dogs. Gonadectomy led to a significant decrease in catalase concentration in gonadectomized dogs compared with other study groups. Malondialdehyde levels increased significantly in gonadectomized dogs compared with other groups. Melatonin administration before gonadectomy led to decreased malondialdehyde concentration in gonadectomized and intact dogs compared to the control group. Cortisol concentration increased significantly in gonadectomized dogs compared to the control dogs. Serotonin levels decreased in gonadectomized dogs, but melatonin treatment increased serotonin concentration in gonadectomized and intact dogs. Melatonin treatment before gonadectomy suppressed oxidative stress and the cortisol but increased serotonin level.

Effects of epididymal sperm recovery methods on fresh and frozen-thawed sperm characteristics in dogs.

The cauda epididymis holds a collectible source of fertile spermatozoa in cases of obstructive azoospermia, sudden death, and after elective or emergency castration. The current study was conducted to compare three different epidydimal sperm collection methods (Percutaneous epididymal sperm aspiration (PESA), microsurgical epididymal sperm aspiration (MESA) and retrograde epididymal wash (EW)) in the dog. Fifteen large-breed adult dogs were applied for comparing the PESA (left testicles) with MESA (right testicles) techniques, while five dogs were used for evaluation of MESA (left testicles) versus EW (right testicles). The recovered sperm cells from MESA and EW were subjected to cryopreservation. Total sperm recovery, level of blood contamination and sperm quality markers (viability, morphology, plasma and acrosome membrane integrity, DNA fragmentation, and metabolic activity) were evaluated for fresh and frozen-thawed spermatozoa. We showed that the collection of epididymal sperm cells through the PESA method resulted in lower total sperm recovery and significantly reduced fresh sperm kinematic and quality measures. While, both MESA and EW procedures resulted in a high number of intact epididymal spermatozoa with appropriate cryo-tolerance potential. In conclusion, EW and MESA methods provide high-quality epidydimal spermatozoa with high cryopreservation potential in domestic dogs.

The effects of melatonin treatment on oxidative stress induced by ovariohysterectomy in dogs.

BACKGROUND: As one of the most common surgeries performed in veterinary medicine, ovariohysterectomy (OHE) can induce oxidative stress in dogs. The antioxidant properties of melatonin have been confirmed in various studies. This study aimed to investigate the effects of melatonin administration on oxidative stress in dogs before and after OHE. In this study, 25 mature female intact dogs were selected and randomly divided into five equal groups: Melatonin (melatonin, no surgery), OHE (no melatonin, surgery), OHE + melatonin (melatonin, surgery), anesthesia+melatonin (melatonin, sham surgery), and control (no melatonin, no surgery) groups. Melatonin (0.3 mg/Kg/day, p.o.) was administrated to the dogs in the melatonin, OHE + melatonin, and anesthesia+melatonin groups on days - 1, 0, 1, 2, and 3 (day 0 = OHE). Blood sampling was performed on days - 1, 1, 3, and 5 of the study. Blood samples were immediately transferred to the laboratory and sera were separated and stored at - 20 °C. Superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and malondialdehyde (MDA) concentrations were measured with commercial kits. RESULTS: The levels of SOD, GPX and CAT were significantly higher in the melatonin and anesthesia+melatonin groups compared to those of the control group at days 3 and 5. The level of antioxidant enzymes significantly decreased in the OHE group compared to that of other groups at days 3 and 5. The administration of melatonin increased the level of antioxidant enzymes in ovariohysterectomized dogs. Ovariohysterectomy significantly increased the concentration of MDA in comparison to that of other groups at day 3. Melatonin administration significantly decreased the level of MDA in melatonin, anesthetized, and ovariohysterectomized dogs at day 3. CONCLUSIONS: Administration of melatonin on day - 1, 0, 1, 2 and 3 modulate the oxidative stress induced by OHE in dogs by increasing antioxidant enzymes concentration and decreasing MDA levels.

A descriptive angiographic study of the uterine arteries during pregnancy, the postpartum period and CEH/pyometra in bitches.

The aim of this descriptive study was to monitor the changes in uterine arteries during pregnancy, postpartum period and pyometra in bitches using angiography. Fifteen uteri of mixed breed bitches on days 24, 30, 33, 40, 43, 47, 50 and 56 of pregnancy and weeks 1, 2, 3, 4 and 7-8 of postpartum and two CEH/pyometra bitches were examined after ovariohysterectomy. The results showed that with the onset of normal pregnancy and in about 30 ± 1 days of gestation, anastomoses begin to form between the left and right middle uterine arteries, developing during the next days and continuing until 4 weeks postpartum. On 4th week after parturition, when physiologic changes occur and the uterus returns to non-pregnant conditions, these anastomoses begin to degenerate, and they completely disappear approximately on the 7th-8th week after parturition. Similarly, in CEH/pyometra bitches, anastomoses were formed between left and right median uterine arteries. These findings can be considered as a part of the physiological changes of the uterus and its vessels during pregnancy and postpartum periods and could affect the results and interpretation of relevant findings.