Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.     

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.     

Effect of Salinity on Growth and Survival in Juvenile Opossum Pipefish,Microphis brachyurus,in Culture Conditions

Natural populations of the opossum pipefish, Microphis brachyurus, are affected in Mexico by anthropogenic alteration of their habitat and unregulated fishing as this species is traded as an aquarium fish without formal records as occurs with other syngnathids around the world. M. brachyurus is an estuarine fish adapted to salinity fluctuations. The aim of this study was to examine the effect on growth, condition, and survival of juvenile pipefish cultured for 4 wk at salinities of 0, 8, and 16 g/L. Pipefish were fed enriched Artemia nauplii maintaining a ration rate of 14% body weight per day (dry weight Artemia: wet weight fish). Pipefish cultured at 16 g/L showed lower survival (40%) than those at 0 and 8 g/L (100 and 96%, respectively), while juveniles cultured in 8 and 16 g/L were heavier (0.393 and 0.388 g, respectively), longer (9.9 cm), and grew faster (1.4) than those in 0 g/L (0.294 g, 9.1 cm, and 0.44, respectively). This study is the first to report that a salinity of 16 g/L compromises M. brachyurus culture potential and a salinity of 0 g/L caused poor growth while the optimal salinity for the species appears to be around 8 g/L.     

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.     

Influence of Gonadotrophin‐Induced First Oestrus on Gilt Fertility

The aim of this study was to determine the association between the oestrous response of pre‐pubertal gilts to gonadotrophin injection or boar exposure and their subsequent farrowing rate and litter size. At 154 days of age, randomly selected pre‐pubertal gilts received an intramuscular injection of 400 IU equine chorionic gonadotrophin plus 200 IU human chorionic gonadotrophin (PG600^®; Merck Animal Health; n = 181). From the remaining pool of animals not treated with hormones, the first gilts showing signs of oestrus were selected to act as controls (n = 201). Boar exposure began at 155 days of age for both groups, and gilts were bred at a weight of approximately 130 kg. Comparisons were made between PG600^®‐treated gilts exhibiting oestrus or not within 7 days post‐injection (early and late responders, respectively) and control gilts exhibiting oestrus or not within 30 days after beginning of boar exposure (select and non‐select control gilts, respectively). By 162 days, oestrus was detected in 67.5% of PG600^®‐treated gilts compared with 5.7% of control gilts (p < 0.0001). The proportion of animals observed in oestrus at least three times before breeding was greater for select control gilts compared with early and late responder PG600^®‐treated gilts (p ≤ 0.001). There were no significant differences in farrowing rate and litter size between the four treatment groups. These data indicate that PG600^® is an effective tool to induce an earlier oestrus in gilts, that subsequent farrowing rate and born alive litter size compare favourably to that of select gilts and that gilts failing to respond promptly to hormonal stimulation do not exhibit compromised fertility.     

Nitrogen mineralization and CO2 and N2O emissions in a sandy soil amended with original or acidified pig slurries or with the relative fractions

The following six pig slurries obtained after acidification and/or solid/liquid separation were used in the research: original (S) and acidified (AS) pig slurry, nonacidified (LF) and acidified (ALF) pig slurry liquid fraction, and nonacidified (SF) and acidified (ASF) pig slurry solid fraction. Laboratory incubations were performed to assess the effect of the application of these slurries on N mineralization and CO₂ and N₂O emissions from a sandy soil. Acidification maintained higher NH₄ ⁺-N contents in soil particularly in the ALF-treated soil where NH₄ ⁺-N contents were two times higher than in LF-treated soil during the 55–171-day interval. At the end of the incubation (171 days), 32.9 and 24.2 mg N kg⁻¹ dry soil were mineralized in the ASF- and SF-treated soils, respectively, but no mineralization occurred in LF- and S-treated soils, although acidification decreased N immobilization in ALF- (−25.3 mg N kg⁻¹ soil) and AS- (−12.7 mg N kg⁻¹ soil) compared to LF- (−34.4 mg N kg⁻¹ soil) and S-treated (−18.6 mg N kg⁻¹ soil) soils, respectively. Most of the dissolved CO₂ was lost during the acidification process. More than 90% of the applied C in the LF-treated soil was lost during the incubation, indicating a high availability of the added organic compounds. Nitrous oxide emissions occurred only after day 12 and at a lower rate in soils treated with acidified than nonacidified slurries. However, during the first 61 days of incubation, 1,157 μg N kg⁻¹ soil was lost as N₂O in the AS-treated soil and only 937 in the S-treated soil.     

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.     

Laboratory study of the effects of two nitrification inhibitors on greenhouse gas emissions from a slurry-treated arable soil: impact of diurnal temperature cycle

An automated laboratory soil incubation system enabled the effects on gaseous emissions from a soil to be quantified accurately, when amended with slurry plus a nitrification inhibitor: dicyandiamide (DCD), or 3,4-dimethylpyrazole phosphate (DMPP). Nitrification inhibitors applied with slurry under simulated Portuguese conditions were very efficient in reducing N₂O emission, and did not increase CH₄ emissions significantly, when the soil was predominantly aerobic. The inhibitors were also indirectly effective in reducing N₂O emissions due to denitrification during a subsequent anaerobic phase. All gaseous emissions followed strong diurnal patterns that were positively correlated with soil temperature and obeyed a Q₁₀=2 relationship. The widespread use of DCD and DMPP inhibitors with slurry applied to Portuguese soils could have the potential to reduce N₂O emissions from this source by ten- to 20-fold.