Immunohistochemical localization of insulin-like growth factor-I receptor (IGF-IR) in the developing and mature rat testes

It has been suggested that insulin-like growth factor-I (IGF-I) plays an important role in the regulation of spermatogenesis in the testes. Its signal is mediated predominantly by the IGF-I receptor (IGF-IR). Signalling through IGF-IR has been shown to have a potent survival function. IGF-IR, a transmembrane tyrosine kinase, is widely expressed across many cell types. In this study, we demonstrated the distribution of IGF-IR in testes of differently aged rats. Anti-IGF-IR is a rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus of the IGF-IR of human origin. Testicular specimens were fixed in Bouin's solution and embedded in paraffin. The paraffin-embedded sections were processed for standard immunohistochemistry by the labelled streptavidin-biotin technique. At postnatal day 19, IGF-IR immunoreactivity was seen moderately in spermatogonia, and slightly both in leptotene and zygotene primary spermatocytes. At postnatal day 35, immunoreactivity was seen slightly both in the pachytene primary spermatocytes and Leydig cells. Although there was intense immunoreactivity in the Leydig cells and in the elongated spermatids on days 50 and 70, the intensity of reaction was decreased in the elongated spermatids in the 10th month. Our results suggest that IGF-IR may play significant roles in testicular function and germ cell development.     

The characterization of cycad palm toxicosis and treatment effects in 130 dogs

The purpose of this retrospective study is to characterize the clinical signs, laboratory values, treatment modalities and mortality outcomes related to cycad palm toxicosis in dogs and to identify medical interventions that potentially correlate with mortality. Dogs with confirmed cycad palm ingestion were identified by reviewing medical records from two private veterinary referral centres for key terms and phrases indicative of cycad palm toxicosis. Of 130 dogs included, 107 (82.3%) presented with clinical signs. A total of 2 (1.5%) died and 14 (10.8%) were euthanized. Diarrhea, lethargy and mortality were all associated with an elevated initial alanine transaminase (ALT) concentration. Overall mortality was 12.3%, with a significantly higher proportion of dogs with an ALT concentration <125 U/L surviving compared to dogs with an elevated ALT concentration. Treatment with activated charcoal reduced the odds of death among all dogs by 82% and was even more protective among dogs with an elevated initial ALT concentration. Among dogs that had an initial platelet count performed, the presence of thrombocytopenia (<200,000/μL) was a negative prognostic indicator and was associated with a significant increase in mortality. Given the high mortality rates reported for cycad palm toxicosis, it is imperative to identify early clinical indicators of mortality as well as treatments that reduce mortality.     

Modulation of Ovarian Function in Female Dogs Immunized with Bovine Luteinizing Hormone Receptor

Adult female dogs were immunized with 0.5 mg bovine luteinizing hormone receptor (LH-R) encapsulated in a silastic subdermal implant and subsequently with four intramuscular booster injections of 0.1 mg LH-R each. Circulating LH-R antibody was detected in the sera 3 weeks post-implant. The appearance of LH-R antibody was associated with a decline in the serum progesterone concentrations to a range of 0–0.5 ng/ml until day 365 in the immunized dogs in comparison with a range of 5–10 ng in the control animals, suggesting a lack of ovulation and corpus luteum function in immunized dogs. The immunized dogs did not show signs of `standing heat' and failed to ovulate when induced by LH-RH challenge. Serum oestradiol levels, however, remained in the range of 30–40 pg/ml in both the immunized and the control dogs. With the decline in the antibody titres, the hormonal profile and vaginal cytology returned to a fertile state and the dogs exhibited signs of `standing heat', as well as vaginal bleeding. Dogs immunized with LH-R did not show any serious metabolic, local or systemic adverse effects. The hypothalamic–pituitary gonadal axis remained intact as indicated by little difference in pituitary LH levels between control and immunized animals, and by the release of LH by LH-RH challenge. These studies demonstrate that active immunization of female dogs with LH-R could immunomodulate ovarian function to cause a reversible state of infertility. It may be postulated that, due to extensive interspecies homology, a recombinant LH receptor-based immunocontraceptive vaccine may also be effective in other vertebrates.     

Evaluation of a citrate-based anticoagulant with platelet inhibitory activity for feline blood cell Counts

Abstract: Aggregation of feline platelets in vitro results in difficulty assessing platelet number. A citrate-based anticoagulant containing the platelet inhibitors theophylline, adenosine, and dipyridamole (CTAD; Diatube-H, Becton Dickinson, Oxford, UK) has been developed for use in human platelet studies and heparin assays. To evaluate the efficacy of CTAD in reducing platelet aggregation in feline blood samples, aliquots of blood from 51 cats were anticoagulated with EDTA, CTAD, and for 12 samples, citrate solution. Samples preserved in CTAD had significantly higher (P ≤ .001) platelet counts, as determined by an impedance counter, hemacy-tometer, and smear estimation, than samples preserved in EDTA. In addition, subjective assessment of blood smears showed significantly fewer platelet aggregates (P<.001) in CTAD-treated samples compared with EDTA samples. Although values were similar, automated platelet counts and smear estimates of platelet number were significantly higher (P < .05) and platelet aggregation was significantly less (P < .05) in CTAD samples than in citrate samples. These results suggest that the platelet inhibitory activity of CTAD reduced feline platelet aggregation. Automated total WBC counts in CTAD samples were significantly lower (P<.001) than automated counts in EDTA samples but were similar to manual WBC counts in EDTA samples. Differences in both platelet and WBC counts between CTAD and EDTA or citrate samples were clinically relevant. Mean platelet volume and MCV were significantly lower (P< .05) in CTAD samples than in EDTA samples. No effect was seen on cell morphology or staining characteristics. The anticoagulant CTAD offers an advantage over both EDTA and citrate for feline hematologic analysis, by decreasing pseudothrombocytopenia and pseudoleukocytosis.     

Immunolocalization of Melatonin and Follicle‐Stimulating Hormone Receptors in Caprine Ovaries and their Effects During in vitro Development of Isolated Pre‐Antral Follicles

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles.     

Effect of Daily Food Supplementation with Essential Fatty Acids on Canine Semen Quality

Polyunsaturated fatty acids are important membrane components that influence membrane integrity and fluidity. In the present study, the effect of oral supplementation for 60 days with essential fatty acids (omega 3, 6 and 9) and vitamin E on canine semen quality was evaluated. Sixteen dogs were selected for the experiment; eight were used as the control group and eight received the fatty acid supplemented diet for 60 days. Semen samples were taken every 15 days during the entire experimental period and were analyzed for volume (ml), motility (%), vigour (0–5), concentration (×10⁶/ml), morphology of spermatozoa (%), plasma membrane integrity (%; using the hyposmotic swelling test) and thermoresistance (motility and vigour after 4 h at 38°C). We concluded that, daily supplementation with omega 3, omega 6 and omega 9 fatty acids, together with vitamin E, for a period of 60 days, significantly increased the semen volume of the treated group after 15 days of supplementation; the vigour and concentration of spermatozoa were superior after the first month of supplementation, while the percentage of morphologically abnormal spermatozoa decreased and the cells were protected against thermal stress.     

Immunohistochemical investigation of cerebellum in dogs infected with canine distemper virus

The cerebella of 21 dogs with canine distemper virus (CDV) infection and four normal dogs were examined histopathologically and immunohistochemically. Cerebella of CDV-infected dogs showed nonsuppurative demyelinating encephalomyelitis, classified as acute, subacute or chronic. Immunolocalisation of CDV antigen also confirmed the infection. Tissues were examined for co-localisation of the CDV antigen with either an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or an oligodendrocyte-specific marker, galactocerebroside (GalC). Immunoreactive cells were counted in demyelinating areas of the white matter. The number of astrocytes (GFAP positive) was significantly (p < 0.05) higher in CDV-infected dogs compared to controls. In contrast, the number of oligodendrocytes (GalC positive) was significantly (p < 0.001) lower in CDV-infected dogs and was much lower in chronic cases (p < 0.05). Approximately 41% of astrocytes and 17% of oligodendrocytes were immunoreactive for CDV. The ratio of CDV-infected oligodendrocytes and astrocytes remained almost constant during the progression of the disease (P > 0.05). In conclusion, CDV infects both astrocytes and oligodendrocytes. The gradual loss of oligodendrocytes is most likely responsible for the progressive demyelination in CDV infection. Astrocytosis in CDV infection should be further investigated if it occurs to stimulate oligodendrocytes for myelin production to compensate for the loss or to induce oligodendrocyte degeneration.     

Effects of dietary <Emphasis Type="SmallCaps">l</Emphasis>-carnitine and chromium picolinate supplementations on performance and some serum parameters in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

An experiment was conducted to determine the effects of supplemental dietary l-carnitine, chromium picolinate (Cr-Pic) and their combination on growth performance and serum total protein, cholesterol, triglyceride and glucose of rainbow trout (Oncorhynchus mykiss). A total of 276 rainbow trout were randomly allocated to four groups. Fish (average initial body weight = 151 ± 1.69 g) were fed a basal diet without supplemental l-carnitine and chromium picolinate in the control group. The basal diet was supplemented with either 500 mg/kg l-carnitine, 1.6 mg/kg chromium picolinate or 500 mg/kg l-carnitine plus 1.6 mg/kg chromium picolinate for experimental groups 1 (C), 2 (Cr-Pic) and 3 (C + Cr-Pic), respectively. Fish were fed twice a day to apparent satiation for 58 days. Weight gain, growth rate, feed consumption and feed conversion ratio (feed/gain) were calculated for the whole period. At the end of the study, six fish were chosen randomly from each tank to represent the experiment and frozen at −20°C for subsequent dorsal muscle analysis. The results showed no significant differences in final body weight, weight gain or feed conversion ratio among groups. There were also no significant differences in serum total protein, cholesterol, triglyceride or glucose concentrations among groups. Significantly higher lipid concentration of dorsal muscle was observed in experimental groups 1 and 3. In conclusion, supplemental dietary l-carnitine, chromium picolinate and their combination have no beneficial effects on improving growth performance and feed conversion ratio in rainbow trout. However, dietary l-carnitine slightly increased lipid concentration in dorsal muscle of rainbow trout.     

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200‐230 μm) were isolated and cultured in α‐minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α‐MEM: antioxidant‐free medium) or α‐MEM also added by transferrin, selenium and ascorbic acid (α‐MEM+: with antioxidant) or α‐MEM added by PCA (56.25; 112.5; 225; 450; or 900 μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 μg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 μg/ml PCA, compared to the α‐MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 μm) were similar (p > .05) among all treatments containing PCA and α‐MEM+, and those were superior (p < .05) than α‐MEM, except for 450 μg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α‐MEM+ than in α‐MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α‐MEM+ and 56.25 μg/ml PCA. In conclusion, PCA at 56.25 μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.