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.

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.

Changes in thyroid hormones, leptin, ghrelin and, galanin following oral melatonin administration in intact and castrated dogs: a preliminary study.

BACKGROUND: Melatonin regulates metabolism and metabolism related hormones in mammalians. Castration has some adverse effects on the metabolic hormones of dog. This study was conducted to determine the effects of oral melatonin administration on metabolic hormones, as well as to compare changes of these hormones after administration of melatonin in castrated and intact dogs. Twenty healthy mixed breed mature male dogs were divided randomly into four groups (n = 5): melatonin (3 mg/10 kg(, castrated, castrated and melatonin treated, and negative control. Blood sample was collected from jugular vein weekly for 1 month. RESULTS: T3 and T4 hormones had a significant decrease within 1 month following administration of melatonin. No significant change was observed in concentration of FT3 and FT4 hormones. Leptin and ghrelin hormones also had a significant decrease in this period. Leptin and ghrelin had a more significant decrease in "non-castrated and melatonin treated" group compared to "castrated and melatonin treated" group. Galanin had a significant decrease but this neurotransmitter had no significant change in "non-castrated and melatonin treated" group in comparison to "castrated and melatonin treated" group. CONCLUSIONS: It seems that daily administration of melatonin capsule in all dogs can probably decrease concentration of T3 and T4 hormones and balance other metabolic hormones following castration. METHODS: The dogs underwent castration, melatonin treatment and blood sampling.

Changes in specific serum biomarkers during the induction of prostatic hyperplasia in dogs.

BACKGROUND: Prostatic hyperplasia (PH) is one of the most important disorders in intact dogs. In this study, we aimed to induce PH experimentally using the combination of testosterone and estrogen and evaluate important factors associated with this disease. RESULTS: The results showed that in the induction group, prostate volume and prostate specific antigen (PSA) concentration increased significantly on day 21 onwards compared to those of the control group. Canine prostatic specific esterase (CPSE) and dihydrotestosterone (DHT) concentrations increased significantly on day 42 onwards while the testosterone levels increased on day 63. In addition, prostatic acid phosphatase (PAP) concentration did not change significantly in the control and induction groups. Biochemistry profiles and hematologic factors were measured for monitoring the function of liver and kidney, and there were no adverse effects following the induction of PH. CONCLUSIONS: It seems that testosterone and estrogen administration led to prostatic hyperplasia during 2 months. Investigating the size of the prostate, accompanied by prostate markers including CPSE, PSA, DHT, and testosterone, is helpful for the PH diagnosis. However, further studies should be carried out on PAP.

MicroRNA expression in infertile men: its alterations and effects.

Infertility is an important reproductive health problem, and male infertility is especially important in more than half of infertility cases. Due to the importance of genetic factors in this condition, analysis of semen alone is not enough to recognize men with idiopathic infertility. A molecular non-invasive investigation is necessary to gain valuable information. Currently, microRNAs (miRNAs) are being used as non-invasive diagnostic biomarkers. miRNAs, single-stranded non-coding RNA molecules, act as post-transcriptional gene silencing regulators either by inhibition or repression of translation. Changes in the regulation of miRNAs have been investigated in several different types of male infertility, therefore the biological role of miRNA and gene targets has been defined. The purpose of this study was to review recent research on the altered expression of miRNA in semen, sperm, and testicular biopsy samples in infertile males with different types of unexplained infertility. Changes in miRNA regulation were investigated using microarray and the miRNA levels were confirmed by real-time qRT-PCR. This review explains why creating a non-invasive diagnostic method for male infertility is necessary and how changes in miRNA expression can be used as new diagnostic biomarkers in patients with differing spermatogenic and histopathologic injury.

Changes in the oxidative stress factors and inflammatory proteins following the treatment of BPH-induced dogs with an anti-proliferative agent called tadalafil.

BACKGROUND: Finding a medical treatment which can combat cell proliferation and relax smooth muscles in canine benign prostatic hyperplasia (BPH) appears to be imperative. AIMS: This study aimed to evaluate the oxidative stress and inflammatory proteins following the treatment of dogs induced for BPH with an anti-proliferative agent called tadalafil. MATERIALS AND METHODS: Twenty-five adult intact male dogs were randomly designated into five groups (n = 5): Control group was not induced for BPH and not treated with tadalafil; dogs induced for BPH by testosterone enanthate and estradiol benzoate and treated with tadalafil (5 mg/day P.O.); dogs which received tadalafil (5 mg/day P.O.); dogs induced for BPH and treated with castration; and dogs induced for BPH. Oxidative stress factors (glutathione peroxidase [GPX], superoxide dismutase [SOD], catalase) and inflammatory proteins (haptoglobin, serum amyloid A [SAA], malondialdehyde [MDA]) were measured in the blood serum for four sequential weeks. RESULTS: Glutathione peroxidase and SOD serum levels declined in dogs in the BPH-induced group compared to those in the control group. Those levels diminished in BPH-induced castrated and tadalafil-treated groups. The changes in the GPX and SOD serum concentrations were not significant between the BPH-induced castrated group and BPH-induced tadalafil-treated group. Moreover, MDA concentration increased slightly in groups with BPH and groups which were castrated. Generally, however, there were no significant differences in the MDA serum concentrations between other groups. Haptoglobin and SAA concentrations increased in BPH-castrated group. Also, the differences in haptoglobin and SAA were not significant between the groups. CONCLUSION: Tadalafil could not control oxidative stress and inflammatory mediators which happened during BPH in dogs.

Canine and feline foetal fluids: Volume, hormonal and biochemical characterization during pregnancy.

BACKGROUND AND OBJECTIVES: This study aimed to evaluate the volume, the concentration of steroid hormones, and biochemical composition of the foetal fluids at different gestational ages in dogs and cats. METHODS: Following the ovariohysterectomy, the allantoic and amniotic fluid samples were collected from pregnant bitches and queens and were assigned to different groups according to their gestational age. RESULTS: The canine and feline allantoic fluid volume increased during pregnancy, reached its maximum values on days 40-49 and then decreased. The canine and feline amniotic fluid volume increased steadily by the last days of pregnancy. In spite of significant changes of sex hormones in the foetal fluids, their concentration and ratios were not significantly different between male and female fetuses. The canine amniotic cortisol concentration increased until days 40-49 and decreased significantly afterwards. The maximum cortisol concentrations in the feline allantoic and amniotic fluids were observed on days 50-60 and 40-49, respectively. During the canine pregnancy, the concentrations of calcium, phosphorus, chloride, sodium, triglyceride, cholesterol, total protein, albumin and the activities of aminotransferase (AST), alkaline phosphatase (ALP), amylase and gamma-glutamyl transferase (GGT) in the amniotic fluid were higher than the allantoic fluid. The magnesium, potassium, lactate dehydrogenase (LDH) activity, creatine and lipase were higher in the allantoic fluid. In the feline allantoic fluid, potassium, magnesium, phosphorus, creatinine, albumin and glucose concentrations and the activities of creatine kinase (CK), GGT, LDH and lipase were higher. The ALP, AST activities, sodium and calcium concentrations were higher in the amniotic fluid (p < 0.05). CONCLUSION: Volume of foetal fluids was determined in dogs and cats. Concentration of sex hormones did not different between male and female fetuses.

The effects of melatonin on the concentrations of inflammatory cytokines and proteins, serotonin, cortisol and melatonin in ovariohysterectomised female dogs.

BACKGROUND: Ovariohysterectomy (OHE) induces inflammation and stress in female dogs. The anti-inflammatory effects of melatonin have been reported in several studies. OBJECTIVES: The goal of this study was to assess the effects of melatonin on the concentrations of melatonin, cortisol, serotonin, α-1-acid glycoprotein (AGP), serum amyloid A (SAA), c-reactive protein (CRP), interleukin-10 (IL-10), interleukin-8 (IL-8), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) before and after OHE. METHODS: The total number of animals was 25 and aligned in 5 groups. Fifteen dogs were divided into three groups (n = 5): melatonin, melatonin+anaesthesia and melatonin+OHE and received melatonin (0.3 mg/kg, p.o.) on days -1, 0, 1, 2 and 3. Ten dogs were assigned to the control and OHE groups (n = 5) without melatonin treatment. OHE and anaesthesia were performed on day 0. Blood samples were obtained via jugular vein on days -1, 1, 3 and 5. RESULTS: Melatonin and serotonin concentrations significantly increased in the melatonin, melatonin+OHE and melatonin+anaesthesia groups compared with the control group, while cortisol concentration decreased in the melatonin+OHE group compared with the OHE group. The concentrations of acute-phase proteins (APPs) and inflammatory cytokines significantly increased after OHE. The CRP, SAA and IL-10 concentrations decreased significantly in the melatonin+OHE group compared with the OHE group. The concentrations of cortisol, APPs and proinflammatory cytokines increased significantly in the melatonin+anaesthesia group compared with the melatonin group. CONCLUSIONS: The oral administration of melatonin before and after OHE help controlling the high levels of inflammatory APPs, cytokines and cortisol induced by OHE in female dogs.

Ultrasonographic and macroscopic study of pregnancy in golden hamster.

BACKGROUND: Hamster is widely used as an experimental model in the study of reproductive system. However, pregnancy diagnosis and aging always have been a challenge. ultrasonography have been used in diagnosis of pregnancy in some small laboratory animals, such as rabbits, rats, and mice. Current study describes use of trans-abdominal ultrasonography for pregnancy diagnosis and fetal age estimation in golden hamster. Furthermore, a macroscopic examination was performed to evaluate the embryonic vesicle diameter, crown-rump length, and fetal head diameter. Ten adult female golden hamsters were selected and maintained under controlled light conditions (14 h light/10 h darkness). The estrous cycle was synchronized using eCG and hCG. During estrous (18 h after hCG injection), the hamsters were naturally mated. After seven days of mating, the hamsters were examined daily for pregnancy diagnosis and aging with an ultrasound scanner equipped with an 8.5-MHZ linear probe. On each day of the experiment, at least one of the pregnant hamsters was euthanized and dissected for macroscopic fetal measurements using a digital caliper. RESULTS: The gestational sac and crown-rump length were identified and measured by ultrasonographicly on day 7 of pregnancy and head could be visible after day 10 of gestation. Statistical analysis revealed that the ultrasound estimation of gestational age was significantly correlated with the actual age of the fetus (r = 0.98; p < 0.05). CONCLUSIONS: Real-time ultrasound can be used for the diagnosis of pregnancy and estimation of fetal age in golden hamster from day 7 of gestation.

Echotexture Analysis of Prostate Parenchyma for Detection of Benign Prostatic Hyperplasia in Dogs.

Ultrasonography is one of the most common methods for the diagnosis of prostate disorders, such as benign prostatic hyperplasia (BPH), in dogs. Changes in the echotexture are one of the indicators used to diagnose prostate disorders. The purpose of this study was to investigate the changes occurred in the dogs' prostate echotexture during the induction of BPH using image analysis. Twenty sexually mature male intact mixed-breed dogs were selected and divided randomly into control and BPH-induced groups. BPH was induced using testosterone and estrogen injections for 63 days. The ultrasound imaging of the dogs' prostate was performed during the induction of BPH on days 0, 21, 42, and 63. The echotexture of the prostate parenchyma was analyzed using the Image J software. Then, the changes in the echotexture and its correlation and linear regression with the prostate volume and canine prostate specific esterase (CPSE) concentration were evaluated by statistical tests. The prostate parenchyma echotexture did not show any significant changes during the induction of BPH and in comparison with that of the control group. While prostate volume and CPSE concentration increased significantly, indicating that BPH was induced in the dogs. There was no significant correlation and linear regression between the prostate echotexture and prostate volume or between the CPSE concentration and prostate echotexture. According to the results, the alteration in the prostate parenchymal echotexture did not occur in the early stages of induced BPH, but significant changes occurred in the prostate volume and CPSE concentration during those early stages.

Direct endoscopic lavage and biopsy sampling and evaluation of uterine microflora in various stages of the canine estrous cycle.

The microbial population of the uterus fluctuates during the estrous cycle. Microflora of uterus may affect the establishment and maintenance of pregnancy in bitches. The endoscopic samples obtained from the vagina and uterus of 20 female adult mixedbreed dogs. The uterine lavage samples were prepared for cytology, bacterial (aerobic and anaerobic) and fungal cultures. Uterine tissue samples were evaluated for the presence of E. coli by the polymerase chain reaction. The pure growth of bacteria was observed in seven plates out of the nineteen cultured samples (36.84%) and five Gram-negative and two Gram-positive bacteria were detected. The highest number of isolated bacteria was related to the samples of the diestrus and anestrus stages of the estrous cycle, while the lowest number of bacteria was observed in the samples of the estrous stage. Moreover, Citrobacter spp. was the most frequent group of isolated bacteria. The neutrophils were detected in the cytology of uterine samples. The fungi growth was observed in three uterine samples. Cladosporium and Penicillium isolated from the samples were related to the estrus stage, and yeast was grown in diestrus samples. The 16srRNA gene existed in all of the estrous uterine samples in which the bacterial culture was negative. However, the presence of this gene was proven in two samples (33.30%) of negative bacterial culture samples from the diestrus and anestrus stages. In conclusion, the normal bitches' uteri were infected with various bacteria in estrus, diestrus and anestrus stages of the estrous cycle, and it could coincide with the fungi infection.

The detection of canine anti-sperm antibody following parenteral immunization of bitches against homogenized whole sperm.

BACKGROUNDS: The development of a canine-specific method of immunocontraception is one of the non-invasive controlling strategies for humanely decreasing the dog population. This study was aimed to investigate the potential of whole sperm in stimulating the immune system and producing specific anti-sperm antibodies (ASAs) in female dogs. Mature, mixed-breed bitches were subcutaneously immunized with high (200 × 106 cells/mL) and low (100 × 106 cells/mL) doses of sperm vaccine, emulsified with Freund's adjuvants. Booster immunizations were given at weeks 1, 2, 4, and 6, and serum samples were collected at days 0, 14, 28, 42, 63, and 84 prior to each immunization. Reproductive tract samples, including vaginal and uterine lavages, were also collected by flushing each section with sterile PBS at the end of the experiment. Canine anti-sperm antibody titer and specificity in sera and genital secretions were measured using an enzyme-linked immunosorbent assay technique. RESULTS: Specific anti-sperm antibodies were detected in the serum of both high and low dose groups and were significantly higher than those observed in the controls. A high dose of sperm induced elevated immune responses over the low dose antigen. Immunization with a high dose of sperm increased the level of ASAs in the uterine secretions and vaginal secretions significantly. Higher ASAs were observed to have transduced to the uterine lumen compared to the vagina. CONCLUSIONS: Based on the results obtained in this study, parenteral immunization with whole sperm can induce a high level of specific antibodies in the serum and genital secretions of female dogs and the response would be dose-dependent.

CONTEXTE: Le développement d'une méthode d'immunocontraception spécifique à la race canine constitue l'une des stratégies non invasives pour réduire humainement la population canine. Cette étude a pour objectif d'évaluer le potentiel du sperme entier à stimuler le système immunitaire des femelles et à produire des anticorps anti-sperme spécifiques (ASA) chez ces dernières. Des chiennes matures de race croisée sont immunisées par voir sous-cutanée avec soit de fortes doses (200 millions de cellules/mL) soit de faibles doses (100 millions de cellules/mL) de vaccin constitué de sperme entier émulsifié avec des adjuvants de Freund. Des vaccinations de rappel sont faites aux semaines 1, 2, 4 et 6, et des échantillons sanguins sont prélevés aux jours 0, 14, 28, 42, 63 et 84 avant chaque immunisation. Des échantillons de l'appareil reproducteur, incluant des lavages vaginaux et utérins, sont recueillis par flushing de chaque section avec du PBS stérile à la fin de l'expérimentation. Le titre et la spécificité des anticorps anti-sperme entier canin dans le sérum et les sécrétions génitales ont été mesurés par la technique de dosage ELISA. RÉSULTATS: Des anticorps anti-sperme entier spécifiques ont été détectés dans le sérum des femelles immunisées tant avec de faibles que de fortes doses, et de façon significativement plus élevé que chez le groupe témoin. Une dose forte de sperme entier induit des réponses immunitaires élevées par rapport à l'antigène à faible dose. L'immunisation avec une forte dose de sperme entier augmente de façon significative le niveau d'ASA dans les sécrétions utérines et dans les sécrétions vaginales. On a observé que les ASA ont plus été transduits vers la lumière utérine que vers la lumière vaginale. CONCLUSIONS: Basée sur les résultats obtenus dans la présente étude, l'immunisation parentérale par du sperme entier peut induire un taux élevé d'anticorps spécifiques dans le sérum et le sécrétions génitales de chiennes ; et la réponse serait dose-dépendante.

The Effect of Increased miR-16-1 Levels in Mouse Embryos on Epigenetic Modification, Target Gene Expression, and Developmental Processes.

Changes in microRNA (miRNA) levels are present in numerous diseases. Although these changes are particularly noted in male infertility, little is known about the effects of increased miR-16-1 in sperm from infertile men. In this study, we assessed the effects of increased mir-16-1 expression on the developmental process, epigenetic changes, and target gene expressions. IVF embryos, 6 h after insemination, were divided into three groups: control, control negative (CN), and miR-16-1 harboring plasmid microinjection. The developmental rates of these embryos were recorded after 24, 48, 72, and 96 h of culture. The levels of histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 27 tri-methylation (H3K27me3) were assessed in the 2-cell and blastocyst stages by immunofluorescence staining. Expression profiles of the miR16-1, Bax, Bcl-2, Suz12, and Kmt2a genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). There was a significant decrease from the 8-cell stage to the blastocyst stage of embryo development in the miR-16-1 harboring plasmid microinjection group. We observed substantial reductions in the amounts of H3K4me3 and H3K27me3 in the 2-cell and the blastocyst stages in the miR-16-1 harboring plasmid microinjection group (P ≤ 0.05). The miR-16-1 level in the miRNA group was higher than the control group in the 2-cell and the blastocyst stages. There was a significant increase (P ≤ 0.05) in Bax and decreases in Bcl2, Suz12, and Kmt2a following the injection of the miR-16-1 harboring plasmid. These results suggest that a change in miR-16-1 expression can significantly affect embryo development, epigenetic changes, and target gene expressions.