Veterinary Research Forum

Abstract Taraxacum officinale has been used in Jordan folk medicine to treat male infertility. A recent study has proved a contradictory effect of the whole plant aqueous extract. The aim of the current study was to determine if the leaves of T. officinale have similar anti-fertility activities, and whether this effect is mediated through the regulation of spermatogonial stem cells (SSCs). Fifty adult male rats were divided into five groups. Two groups were gavaged with 1/10 of LD₅₀ of T. officinale whole plant (1.06 g kg^-1 body weight) or leaves (2.30 g kg^-1 body weight) aqueous extract; while two groups were gavaged with 1/20 of LD₅₀ of T. officinale whole plant (2.13 g kg^-1) or leaves (4.60 g kg^-1) extract. The control group received distilled water. Oral administration of T. officinale (whole plant and leaves aqueous extract) caused a significant decrease in testis and seminal vesicle weight, a reduction in serum testosterone concentration, impaired sperm parameters, and a decrease in pregnancy parameters. Testicular histology of treated rats showed structural changes such as hypoplasia of germ cells, reduction in the thickness of germinal epithelium, arrest of spermatogenesis at spermatid stage (late maturation arrest) and reduction in the number of Leydig cells. Gene expression levels of two SSCs markers (GFRα1 and CSF1) responsible for self-renewal were relatively counter-balanced. In conclusion, T. officinale whole plant and leaves aqueous extracts changed the gene expression of two SSCs markers leading to the imbalance between spermatogonia self-renewal and differentiation causing late maturation arrest.

Abstract Leptin, the 16-kDa product of the obese (ob) gene, primarily secreted from adipose tissue, has been implicated to play an important role in the regulation of food intake and energy expenditure. This study investigated protective effect of leptin on trichostatin A-induced apoptotic on in vitro maturation ratio of buffalo oocytes. Ovaries were collected from abattoir and were transported immediately to the laboratory by a thermos flask containing sterile normal saline with antibiotics. Oocytes were aspirated from 2 to 8 mm visible follicles. Oocytes were placed in a culture plate and then incubated at 38.5 ˚C with 5% CO₂ in air for 24 hr. The maturation of oocytes was evaluated under a stereomicroscope. The FITC-Annexin V and propidium iodide staining method was used to detect oocyte apoptosis. In leptin treated groups with 0, 10, 50 and 100 ng mL^-1 and groups that apoptosis was induced, the percentage of oocytes maturation was 77.03, 86.12, 85.08, and 79.89% and 59.96, 56.93 and 51.98, respectively, while the percentage of apoptosis was 8.83, 7.90, 8.58, and 9.39%, and 10.37, 11.57 and 12.03, respectively. Our findings showed that addition of 10 and 50 ng mL^-1 leptin to IVM medium of buffalo oocytes could increase oocyte nuclear maturation, and could decrease oocyte apoptosis when trichostatin A added for inducing apoptosis.

Abstract The present study was conducted to evaluate the effect of fish oil supplementation prior to mating on secondary sex ratio of pups (the proportion of males at birth) in bitches. Sixty five bitches (German Shepherd, n = 35; Husky, n = 30) were enrolled in the study. Bitches (140-150 days post-estrus) were given 2% per dry matter intake palm oil and fish oil in the control (n = 33) and treatment (n = 32) groups, respectively. To induce estrus, bitches were received equine chorionic gonadotropin (eCG) administration (50 IU kg^-1) 30 days after nutritional supplementation followed by human chorionic gonadotropin (hCG) administration (500 IU per dog) seven days later. Bitches were introduced to dogs of the same breed after hCG administration. The weight of bitches was increased over time (p < 0.05), but their weight change was not different between two groups (p > 0.05). The mating rate, pregnancy rate and litter size were not influenced by treatment and breed. Secondary sex ratio was higher in the treatment (105/164; 64.00%) than in the control (68/147; 46.30%) group (p < 0.05; adjusted odds ratio = 2.068). Moreover, secondary sex ratio was higher in Husky bitches (88/141; 62.40%) compared to German Shepherd (85/170; 50.00%; p < 0.05; adjusted odds ratio = 1.661). In conclusion, the present study showed that inclusion of fish oil in the diet of bitches prior to mating could increase the proportion of male pups at birth. In addition, it appears that there might be variation among dog breeds with regard to the sex ratio of offspring.

Abstract In this study the cranial nerves development of H. huso are explained from 1 to 54-days-old (1, 3, 6, 15, 21 and 54 days). Despite all the researches on fish brain, there are no study on nerves evolution on H. huso during their larvae life. For this research 40 samples of larvae H. husowere obtained (from each age, about six samples were selected). The specimens were maintained in fiberglass tank, then histological samples were taken from tissues and stained with hematoxylin and eosin for general histological studies using light microscope. According to the results, on 1 and 3-days-old, no nerve was observed. The terminal nerve and their dendrites were observed around the nasal cavity and the axons projected to different areas in forebrain especially around olfactory bulb diffusely, on 6-day-old fish. Also, olfactory, optic, oculomotor, trochlear, trigeminal, lateral line and vagus nerves were detected on 6-day-old fish, however two parts of lateral line nerve were separated on 54-day-old. Three nerves, profundus, facial and octaval were observed on 54-day-old, however, up to this age, epiphysial nerve was not observed.

Abstract Euphrates turtle is the only soft shell turtle of Iran, and unfortunately is in danger of extinction due to multiple reasons. Imaging techniques, in addition to their importance in diagnosis of injuries to animals, have been used as non-invasive methods to provide normal anatomic views. A few studies have been conducted to understand body structure of the Euphrates turtle. Since there is only general information about the anatomy of turtle limbs, the normal skeleton of the Euphrates limbs was studied. For this purpose four adult Euphrates turtles were used. Digital radiographic examination was performed by computed radiographic (CR) in dorsoventral (DV) and lateral (L) positions. Spiral CT-scanning was done and 3D images of the bones were reconstructed for anatomical evaluation. For skeletal preparation, the skeleton was cleaned by a combination of boiling and mealworm methods and limbs’ bones were examined anatomically. In the present study, simultaneous anatomic, radiographic and CT studies of bones in individual turtles made us possible to describe bones anatomically and provided comparable and complementary conditions to represent the abilities of the radiography and CT for better understanding of the anatomy. Arrangement and the number of carpal and tarsal bones are used in turtles’ classification. Among the studied species, Euphrates turtle carpal and tarsal bones show the most similarities to the Apolone spinifera.

Abstract It has been shown that chronic exposure to cypermethrin (CPM), a pyrethroid pesticide, results in follicular atresia via pathologically affecting angiogenesis, disrupting endocrine potential and enhancing oxidative stress. This study was aimed to uncover the CPM-exposed energy dependent follicular cells apoptosis and to estimate protective effect of vitamin E (VitE) as a potent antioxidant. Thirty six Wistar rats were divided into six groups (n = 6 rats for each group) including; control-sham, CPM-received (CPM, 75 mg kg^-1, intraperitoneally), and CPM and VitE-treated (VitE, 150 mg kg^-1, orally) for 14 and 24 days. The protein biosynthesis of glucose transporter-1 (GLUT-1) and caspase-3 in follicles were estimated by using immuno-histochemical staining at preantral and antral stages. Moreover, the periodic acid Schiff (PAS) staining was performed in order to evaluate the intracytoplasmic carbohydrate ratio in follicular cells and oocyte. Percentages of follicles with GLUT-1, Caspase-3 and PAS-positive cells were compared between groups. Immunohistochemical analyses showed that, VitE significantly up-regulated the GLUT-1 expression and improved the intracytoplasmic carbohydrate supplementation especially at preantral follicles. The cross sections from the CPM-exposed ovaries represented remarkable elevation in percentage of atretic preantral and antral follicles with caspase-3 biosynthesis, which was remarkably (p < 0.05) diminished in VitE co-treated groups. In conclusion, our data showed that VitE by up-regulating of the GLUT-1 biosynthesis improved glucose uptake at follicular cells and oocyte levels that in turn inhibited pro-apoptotic protein caspase-3 biosynthesis.

Abstract Cyclophosphamide (CP) is a drug commonly used to treat neoplastic disease and some autoimmune diseases. It is also a well-known and well-studied teratogen causing a variety of birth defects in fetuses of pregnant women treated with the drug. There are many reports that show the adverse effects of CP can be decreased by use of antioxidant drugs. It appears that, quercetin has antioxidant effect. The aim of this study was prevention or decrease of teratogenicity of CP in fetuses of rats by quercetin. This study was performed on 35 pregnant rats divided into six groups. Control group was received normal saline (5 mL kg^-1, intraperitoneally) and 2-6 groups received a single dose of CP (15 mg kg^-1), a single dose of quercetin (75 or 200 mg kg^-1), CP plus quercetin (75 or 200 mg kg^-1) intraperitoneally at 9^th day of gestation, respectively. Fetuses were collected at 20^th day of gestation and after determination of weight and crown rump length were stained by alizarin red – alcian blue method and skeletal system were examined by stereomicroscope. The results showed that the cleft palate, exencephaly, spina bifida and omphalocele incidence were 55.56%, 27.77%, 33.34% and 11.11%, in fetuses of rat that received only CP, respectively. However, it decreased to 16.00%, 16.00%, 16.00% and 8.00% by quercetin (75 mg kg^-1) and so to 12.90%, 12.90%, 6.45% and 3.28% by quercetin (200 mg kg^-1), respectively. On the basis of results, quercetin significantly can decrease teratogenicity induced by CP.

Abstract The present study was carried out to enhance shelf life of pasteurized cream using Echinophora platyloba essential oil (EEO) and lycopene. For this purpose, two concentrations of EEO (0.10% and 0.50%) and lycopene (20 and 50 ppm) alone and together as combinations were added in pasteurized creams and analyzed for microbial characteristics, sensorial properties and lipid stability during storage at 4 ˚C and 25 ˚C for 14 days. Results of microbial and chemical analyses of experimental pasteurized creams showed that pasteurized creams treated with combinations of the EEO and lycopene in their higher concentrations had the best microbial and chemical properties and the most stability than control during storage (p < 0.05). Results of sensorial evaluation demonstrated that all treatments had favorable overall acceptability, even though, the best sensorial properties were observed in creams with combinations of EEO and lycopene in their lower concentrations. Therefore, based on the results of the present study, application of EEO and lycopene as natural preservatives is especially recommend in high fat dairy products such as butter and cream.

Abstract The receptors 1 and 2 of fibroblast growth factor (FGFR1 and FGFR2, respectively) have been observed in all types of testicular cells. Culture on extracellular matrix (ECM) has been observed to lead to initiation of differentiation in spermatogonial stem cells (SSCs). The present study was carried out to investigate whether FGFR1 and FGFR2 play a role in SSCs differentiation. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The gene expression of THY1, cKIT, FGFR1 and FGFR2 was evaluated using quantitative real-time polymerase chain reaction (PCR). Results related to the gene expression of markers of with undifferentiated (THY1) and differentiated (cKIT) spermatogonia implicated stimulation of self-renewal and differentiation in cells cultured on ECM-coated and uncoated plates, respectively (p < 0.05). Concomitantly, the expression of FGFR2 increased during culture in the ECM group (p < 0.05), whereas it did not change in the control group (p > 0.05). As a result, the gene expression of FGFR2 was greater in the ECM than control group (p < 0.05). Nevertheless, FGFR1 expression did not change during culture in the control and ECM groups (p > 0.05). In conclusion, the present study revealed the potential role of FGFR2 in differentiation of SSCs during culture on ECM.

Abstract This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcusspecies were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.

Abstract This study was carried out to determine the presence and frequency of Anaplasma ovis and Anaplasma marginale in sheep and dairy cattle in West-Azerbaijan province, Iran. A total number of 200 blood samples were randomly collected via the jugular vein from apparently healthy cattle (100) and sheep (100). The extracted DNA from blood cells was screened using genus-specific (Anaplasma spp.) nested-polymerase chain reaction (PCR) based on 16S rRNA gene primer sets. Species-specific PCR was set up using major surface protein 4 (MSP4) gene primer set. None of cattle blood samples were positive for Anaplasma spp. by the first nested PCR. Five samples among the 100 sheep blood samples were both positive in the first nested PCR and A. ovis -specific PCR, based on MSP4 gene. In total, 5.00% of animals were A. ovis positive. This study identified a low prevalence of A. ovis in the blood of apparently healthy sheep in West Azerbaijan province.

Abstract Maternal testosterone has been indicated to affect sex ratio of offspring. The present study was conducted to elucidate the role of androgen receptor in this regard by blockade of androgen receptor using flutamide in female mice. Mice were randomly assigned to two experimental groups. Mice in the control (n = 20) and treatment (n = 20) groups received 8 IU equine chorionic gonadotropin (eCG) followed by human chorionic gonadotropin (hCG) injection (8 IU) 47 hr later. In addition, mice in the control and treatment groups received four injections of ethanol-saline vehicle and flutamide solution (2.50 mg), respectively, started from 1 hr before eCG injection until hCG injection at 12-hr intervals. Conception rate was not different between the treatment (18/20: 90.00%) and control (19/20: 95.00%) groups (p > 0.05). Litter size was higher in the treatment (8.22 ± 0.26) than control (7.21 ± 0.28) group (p < 0.05). Male sex ratio was lower in the flutamide-treated mice (67/148: 45.30%) as compared with the untreated ones (80/137: 58.40%; odds ratio = 1.69; p < 0.05). In conclusion, the results showed that androgen receptor blockade could skew sex ratio of offspring toward females implying that the effect of testosterone on sex ratio might be through binding to androgen receptor. In addition, the blockade of androgen receptor using flutamide appeared to enhance litter size.

Abstract The grass carp (Ctenopharyngodon idella) is a herbivorous, freshwater fish species of the family Cyprinidae, and the only species of the genus Ctenopharyngodon. Neoplasms in fishes are generally less aggressive than neoplasms in mammals and are most commonly discrete, focal and benign neoplasms. A 3-year-old grass carp with a big mass on the vertebrae was referred to the clinic. According to the owner’s statements, the fish had no signs of lethargy, ataxia and abnormal behaviors. The size of the mass was 7 × 6 × 6 cm. It cut hardly with audible sounds. The consistency of the mass was as hard as a cartilage. Microscopic examination revealed numerous irregular crests of hyaline cartilage beneath the skin. According to histopathologic characteristics, chondroma on the vertebrae of grass carp was diagnosed.

Abstract Dipygus is a teratological fetus with a double pelvis, genitals, and extremities. Congenital duplications in cattle are rare. Caudal duplication is more common in sheep and pigs while cranial duplications seem to be predominant in cattle.Asymmetric or parasitic conjoined twins consisting of an incomplete twin (parasite) attached to the body of a fully-developed twin (autosite).This report deals with a male Holstein calf with two extra limbs, in the pelvic region which were directed ventrally between the two normal hind limbs. The extra limbs were completely developed in one side and in other side just a bony mass were observed. So classification has been made as asymmetrical attached twins. The genital system was not affected and just one extra kidney-like structure was found. To the authors’ best knowledge, this is the first report of asymmetrical monocephalus dipygus (tetrapus dibrachius) in a male Holstein calf in Iran.