Effect of Prenatal and Neonatal Anti‐Androgen Flutamide Treatment on Aquaporin 5 Expression in the Adult Porcine Ovary

The growth of ovarian follicles is accompanied by fluid‐filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti‐androgen flutamide influences androgen‐dependent AQP5 expression in pre‐antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post‐natally. The ovaries were collected from flutamide‐treated and non‐treated (control) sexually mature pigs. In pre‐antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre‐antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.     

Production of Oocytes of Nile Tilapia (Oreochromis niloticus) for In Vitro Fertilization via Hormonal Treatments

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin‐releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post‐hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.     

Effects of diazepam, ketamine, and their combination on intraocular pressure in normal dogs

Ketamine has been implicated as causing increases in intraocular pressure. The purpose of this study is to document the effects of ketamine, diazepam, and their combination on intraocular pressure (IOP) in normal, unpremedicated dogs. Random-source dogs were assigned to one of five groups of 10 dogs each: ketamine 5 mg kg^–1 (KET5), ketamine 10 mg kg^–1 (KET10), diazepam 0.5 mg kg^–1 (VAL), ketamine 10 mg kg^–1 with diazepam 0.5 mg kg^–1 (KETVAL), saline 0.1 mL kg^–1 (SAL), all given intravenously. A baseline IOP was measured before injection, immediately after injection, and at 5, 10, 15, and 20 minutes following injection. IOP was increased over baseline immediately after injection in the KET5, KET10, and KETVAL groups; at 5, 10, and 15 minutes in the KET5 group; and at 20 minutes in the KETVAL group. The mean IOP change compared to SAL increased immediately after injection and at 5 minutes in the KET5, KET10, and KETVAL groups; at 10 and 15 minutes in the KET5 group, and at 20 minutes in the KETVAL group. The mean IOP increased up to 5.7, 3.2, and 3.1 mm Hg over mean baseline in the KET5, KET10, and KETVAL groups, respectively. All dogs in the KET5 group and the majority in the KETVAL and KET10 groups had an increase in their IOP over baseline. Ketamine caused a clinically and statistically significant elevation in IOP over baseline and compared to SAL. The concurrent addition of diazepam did not blunt this increase. Ketamine should be avoided in dogs with corneal trauma, glaucoma, or in those undergoing intraocular surgery.     

High Embryonic Recovery Rates with In vivo and Ex vivo Techniques in the Bitch

The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans‐surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.     

Animal‐Level Factors Affecting Ovarian Function in Bos indicus Heifers Treated to Synchronize Ovulation with Intravaginal Progesterone‐Releasing Devices and Oestradiol Benzoate

The primary objective of this study was to investigate the impact of animal‐level factors including energy balance and environmental/management stress, on the ovarian function of Bos indicus heifers treated to synchronize ovulation. Two‐year‐old Brahman (BN) (n = 30) and BN‐cross (n = 34) heifers were randomly allocated to three intravaginal progesterone‐releasing device (IPRD) treatment groups: (i) standard‐dose IPRD [Cue‐Mate^® (CM) 1.56 g; n = 17]; (ii) half‐dose IPRD [0.78 g progesterone (P₄); CM 0.78 g; n = 15]; (iii) half‐dose IPRD + 300 IU equine chorionic gonadotrophin at IPRD removal (CM 0.78 g + G; n = 14); (iv) and a control group, 2× PGF_2α [500 μg prostaglandin F_2α (PGF_2α)] on Day ?16 and ?2 (n = 18). Intravaginal progesterone‐releasing device‐treated heifers received 250 μg PGF_2α at IPRD insertion (Day ?10) and IPRD removal (Day ?2) and 1 mg oestradiol benzoate on Day ?10 and ?1. Heifers were managed in a small feedlot and fed a defined ration. Ovarian function was evaluated by ultrasonography and plasma P₄ throughout the synchronized and return cycles. Energy balance was evaluated using plasma insulin‐like growth factor 1 (IGF‐I) and glucose concentrations. The impact of environmental stressors was evaluated using plasma cortisol concentration. Heifers that had normal ovarian function had significantly higher IGF‐I concentrations at commencement of the experiment (p = 0.008) and significantly higher plasma glucose concentrations at Day ?2 (p = 0.040) and Day 4 (p = 0.043), than heifers with abnormal ovarian function. There was no difference between the mean pre‐ovulatory cortisol concentrations of heifers that ovulated or did not ovulate. However, heifers that ovulated had higher cortisol concentrations at Day 4 (p = 0.056) and 6 (p = 0.026) after ovulation than heifers that did not ovulate.     

Optimization Protocol for Storage of Goldfish (Carassius auratus) Embryos in Chilled State

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0°C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat‐stage goldfish embryos were chilled at 0, 4 or 8°C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8°C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8°C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non‐significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat‐stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 μg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Improved Prediction of Ovulation Time may Increase Pregnancy Rates to Artificial Insemination in Lactating Dairy Cattle

A prospective observational study was conducted in two Australian dairy herds to assess the potential for improving pregnancy rates (proportions of inseminations that result in pregnancy) to artificial insemination (AI) if the time of ovulation could be predicted with more certainty. Herd 1 calved year‐round and inseminations were performed during two periods each day. Herd 2 calved during autumn–winter and inseminations were performed only after the morning milking each day. In both herds, the AI to ovulation interval of enrolled cows was determined by trans‐rectal ovarian ultrasonography approximately 0, 12, 24 and 36 h after AI, and pregnancy was assessed by palpation per rectum 35–56 days after AI. Also, in Herd 1 vaginal electrical resistance (VER) measurements were taken at approximately 0, 12, 24 and 36 h after AI, and in Herd 2 cows were fitted with neck mounted activity meters that monitored cow activity count in 2‐h periods. There was substantial variation in the intervals from AI to ovulation within and between herds (mean ± SD 21.2 ± 10.7, n = 102; 14.7 ± 10.4, n = 100 in herds 1 and 2, respectively). Pregnancy rates were higher for inseminations close to, but preceding, ovulation. Using combined herd data (n = 202), the highest pregnancy rate (50.8%) was observed for inseminations between 0 and 16 h before ovulation, a period in which only a modest proportion of inseminations (31.2%) occurred. In contrast, pregnancy rate was significantly lower (28.7%; risk ratio 0.6; 95% CI 0.4–1.0; p = 0.039) for inseminations between 16 and 32 h before ovulation, a period where the highest proportion of inseminations (53.2%) occurred. Thus pregnancy rates could potentially be improved if a greater proportion of inseminations were conducted shortly before ovulation. In Herd 1, mean VER during the peri‐ovulatory period varied with time from ovulation. Lowest values (mean ± SEM, VER = 64.8 ± 1.2, n = 55) occurred approximately 18 h before ovulation and were significantly lower than measurements approximately 6 h before ovulation (67.4 ± 1.0; n = 73; p = 0.003). Further work is required to determine if VER can be used to identify ovulation time and hence the optimal time to inseminate in individual animals. In Herd 2 a modest proportion of inseminations (26.9%) occurred between 24 and 40 h after the onset of increased cow activity where the highest pregnancy rate (67.9%) was observed, whereas a significantly lower pregnancy rate (42.4%; risk ratio 0.6; 95% CI 0.4–0.9; p = 0.036) was observed for inseminations between 8 and 24 h after the onset of increased cow activity where the highest proportion of inseminations (56.7%) occurred. Thus cow activity monitoring may be useful to identify the optimal time to inseminate cows. Results from this study indicate that improved methods of ovulation prediction may allow better insemination timing relative to ovulation and consequently increased pregnancy rates.