Intrinsic innervation of the ileocaecal junction in the horse: Preliminary study

Reason for performing study: In horses, morpho‐functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. Objectives: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). Methods: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene‐related peptide (CGRP) and neurofilament NF200kDa (NF). Results: Ileal FB‐labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 ± 11% and 68 ± 4%, respectively), cholinergic (60 ± 19% and 82 ± 11%, respectively), NF‐IR (54 ± 9% and 78 ± 21%, respectively), and SP‐IR (about 20% in both the plexuses). CGRP‐IR was expressed only by SMP descending neurons (45 ± 21%). In both the plexuses descending neurons coexpressing nNOS‐and ChAT‐IR were also observed (25 ± 11% and 61 ± 27%, respectively). Conclusions: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. Potential relevance: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.     

Intrinsic innervation of the horse ileum

This paper describes the morphology and distribution of the enteric nervous system (ENS) cells and fibres immunoreactive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP), NF200 kDa (NF200), and S100 protein. The percentages of subclasses of enteric neurons in the total neuronal population were investigated by the use of anti-PGP 9.5 or anti-NSE antibodies.ChAT-IR myenteric plexus (MP) and submucosal plexus (SMP) neurons were 66 ± 7% and 74 ± 15%, respectively, whereas those cells expressing nNOS-IR were 38 ± 7% and 5 ± 1%, respectively. MP and SMP neurons expressing both phenotypes were also present. SP-IR was expressed by 14 ± 13% of MP and 66 ± 8% of SMP neurons whereas CGRP-IR was observed only in the SMP (43 ± 6%). NF200-IR was expressed by 61 ± 15% and 91 ± 10% of the MP and SMP neurons, respectively. The majority of the CGRP-IR SMP neurons expressed also SP-IR. Almost all SP-IR neurons in both the plexuses were cholinergic. The present study quantifies the main neuronal subpopulations of the ENS of the horse ileum; these data might be utilized to understand the neuronal modifications which occur in several gastrointestinal tract disorders.     

Immunohistochemical Characterization of Neurones in the Hypoglossal Nucleus of the Pig

With 4 figures and 1 table In this study, the presence of several neurotransmitters and transmitter synthesizing enzymes was studied in hypoglossal nucleus (HN) of the juvenile (4 months old) female pigs (n = 3). Double‐labeling immunofluorescence revealed neurones expressing cholinacetyltranspherase (ChAT), calcitonin gene‐related peptide (CGRP), nitric oxide synthase (NOS), and somatostatin (SOM). Nerve fibers within HN were ChAT, CGRP, NOS, SOM, substance P (SP), Leu‐5‐enkephalin (Leu‐5‐Enk), ß‐dopamine hydroxylase (DßH), neuropeptide Y (NPY) positive. Virtually all the perikarya contained ChAT, whereas CGRP was present in 47% of the neurones. Nerve cell bodies containing NOS or SOM were only occasionally observed. Immunoreactive nerve fibers were found in a close vicinity of the perikarya, often forming baskets around nerve cell bodies. The results obtained were compared with similar data obtained in other species. The presence of immunoreactive structures, origin of the nerve fibers, and functional significance of the findings are discussed.     

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30–40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB‐positive (FB+) neurons were stained using antibodies against calcitonin gene‐related peptide (CGRP) substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP), nitric oxide synthase (n‐NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu‐5‐enkephalin (Leu‐Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n‐NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu‐Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).     

Co‐expression of PACAP with VIP,SP and CGRP in the Porcine Nodose Ganglion Sensory Neurons

Our previous study revealed the expression of substance P (SP) and calcitonin gene‐related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase‐activating polypeptide‐immunoreactive (PACAP‐IR) porcine nodose perikarya. Co‐expression of these substances was examined using a double‐labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan‐neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right‐side ganglion and 66.5% in the left side, respectively, co‐expressed PACAP‐IR. Moreover, 60.6% of the PACAP‐IR cells in the right‐side ganglion and 62.1% in the left, respectively, co‐expressed VIP. SP‐IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co‐expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.     

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co‐exposure to these compounds could lead to drug–drug (D‐D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision‐cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co‐incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8‐fold upon co‐incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2‐hr co‐incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D‐D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug‐metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

The effect of some cryoprotectants on dromedary camel frozen‐thawed semen

The cryopreserved camel semen is often associated with poor quality and fertility. This study aimed to improve the dromedary frozen semen quality by comparing the efficiency of four cryoprotectant agents (CPAs) on sperm freezability. Semen samples were collected from seven male Maghrabi camels, diluted with Shotor diluent supplemented with glycerol (Sh‐G), dimethyl formamide (DMF, Sh‐DF), dimethyl sulfoxide (DMSO, Sh‐DS) or ethylene glycol (EG, Sh‐EG), all at 6% final concentration, and the samples were subjected to cryopreservation. The results revealed the superiority of Sh‐DF over Sh‐G and Sh‐DS in terms of post‐thaw motility (55.83 ± 2.20 vs. 47.50 ± 4.33 and 45.00 ± 2.89%, respectively), sperm membrane (49.00 ± 0.58, 39.33 ± 3.33 and 42.67 ± 1.45%, respectively) and acrosomal integrities (53.00 ± 0.58, 57.33 ± 0.88 and 52.33 ± 1.45%, respectively). Sh‐EG group showed the lowest post‐thaw motility, plasma membrane and acrosome integrities (12.50 ± 1.44, 22.67 ± 1.45 and 30.67 ± 1.45, respectively). In conclusion, the protocols of dromedary camel semen cryopreservation could be enhanced using 6% DMF as a cryoprotectant agent.     

Effect of different methods for conserving rice grain on in situ ruminal degradation and in vivo nutrient digestion and rumen fermentation in steers

We investigated the effects of different rice conservation techniques on in situ ruminal degradation and in vivo nutrient digestibility and rumen fermentation in steers. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS‐A), or ensiled before crushing (ENS‐B). Six ruminally cannulated steers were used in a replicated 3 × 3 Latin square design with three dietary treatments: diets containing DRY, ENS‐A, or ENS‐B at 36% of the dietary dry matter. The in situ rapidly degradable fraction and effective ruminal degradability were higher for ensiled rice than for DRY, and higher for ENS‐A than for ENS‐B. The ruminal pH was lower and the lactic acid and total volatile acid concentrations were higher for the steers fed ensiled rice than those fed the DRY diet, but a treatment effect was not observed in the comparison between ENS‐A and ENS‐B. The whole‐tract digestibility of crude protein and ether extract was improved when the rice grain was ensiled, but there were no differences in nutrient digestibility between ensiling methods. These results show that ensiling treatment can be a strategy to improve the nutrient value of rice grain, but the ensiling method has little impact on in vivo digestion.     

Expression of neuronal nitric oxide synthase in the pancreas of the sheep

In numerous mammals, nitric oxide (NO) influences the activity of the exocrine and endocrine pancreas. In this study, immunocytochemistry was utilized to investigate the expression of neuronal nitric oxide synthase (nNOS) in the pancreas of sheep. In double immunocytochemical staining, the co-localization of nNOS with vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or substance P (SP) was studied. The presence of nNOS was confined to the intrapancreatic neurones (9.6 +/- 1.3%) as well as to nerve fibres of the endocrine pancreas and intrapancreatic ganglia. nNOS-immunoreactive (IR) neurones were round and oval in shape and predominantly (83.3 +/- 2.6%) belonged to the middle-size group (25-50 mum). Numerous, fine islets supplying nNOS-IR nerve terminals were devoid of VIP, SP or NPY. Moderately numerous, non-varicose nNOS-IR nerve fibres of intrapancreatic ganglia frequently expressed VIP or NPY, but not SP; 2.2 +/- 0.6% of nNOS-IR intrapancreatic neurones displayed lack of VIP, whereas 7.5 +/- 0.8% were VIP-IR. All nNOS-IR neurones were devoid of SP. The frequencies of nNOS-IR/NPY-IR and nNOS-IR/NPY-negative intrapancreatic neurones were 2.2 +/- 0.4% and 6.1 +/- 1.1%, respectively. Comparison with other mammals indicated that nitrergic innervation of the ovine pancreas is species-determined and may be a reflection of the ruminants' digestion specificity. The possible origin of nNOS-IR nerve fibres and functional significance of NO in the pancreas of sheep were discussed.     

Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa

The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO‐PRO‐1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post‐thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Effect of testicular tissue lysate on developmental competence of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section‐wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high‐performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co‐incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum‐containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section‐wise TTL supplements, TR section significantly (p < .01) improved cleavage and morula rate (58.43 ± 7.98 and 36.14 ± 6.89, respectively) followed by TA. In conclusion, present study indicates that IVM, in vitro fertilization and in vitro culture of embryo are improved in the presence of TTL, particularly its TR section. Further study is expected to reveal the principal components of TTL which may prove useful for IVM.     

Morphological assessment of cat kidneys using computed tomography

There are numerous publications about feline renal imaging information; however, none have established reference values for kidney size using computed tomography (CT). This study aimed to determine renal size and shape as well as the morphology of renal‐related structures in clinically normal cats (Felis catus) that underwent CT. Twenty‐seven healthy cats underwent pre‐ and post‐iodinated contrast‐enhanced CT. Most cat (59%) kidneys were located at the same level. The average pre‐contrast dimensions of the left kidney included a width of 2.46 ± 0.28 cm, a length of 3.52 ± 0.44 cm and a height 2.19 ± 0.31 cm, whereas those of the right kidneys were 2.45 ± 0.27 cm, 3.54 ± 0.46 cm and 2.05 ± 0.23 cm, respectively. After contrast enhancement, kidneys were slightly enlarged though not significantly. Additionally, renal length (LK or RK) was compared with second lumbar vertebra (L2) length and abdominal aorta diameter (AO). AO was significantly larger in male cats whereas L2 length appeared longer in male cats, but was not statistically different from the female cats. The LK/L2 and RK/L2 ratios were 2.29 ± 0.23 and 2.36 ± 0.20, respectively, and the LK/AO and RK/AO were 11.72 ± 1.37 and 12.05 ± 1.47, respectively. Renal vessels were examined. The renal vein was obviously larger than the renal artery, and paired renal veins were observed periodically. This study provides CT information about the feline kidney, which may help to establish reference values and information regarding renal structure prior to surgery in practice.     

Topical delivery of diclofenac into and across equine skin from a novel liquid diclofenac epolamine formulation

The aim was to investigate diclofenac delivery into and across equine skin in vitro using Franz diffusion cells from a novel diclofenac epolamine (DIC‐EP; 1.3%) formulation and to compare the results to those of Surpass^® (1% diclofenac sodium liposomal cream) and a 1% aqueous solution of diclofenac sodium. Skin was harvested from the lower legs of Freiberger geldings immediately after slaughter and sliced to a thickness of ~2 mm. Skin samples were divided into two groups [Group 1: 1 year old (n = 2) and Group 2: 6–8 years old (n = 3)]. Cumulative permeation of diclofenac in Groups 1 and 2 after 24 h using diclofenac sodium solution was 1.91 ± 0.27 and 1.76 ± 0.34 μg/cm², respectively. The values for Surpass^® and DIC‐EP were 3.2 ± 0.8/3.3 ± 0.7 μg/cm² and 230 ± 59/89.2 ± 32.5 μg/cm², respectively. Thus, diclofenac permeation from DIC‐EP was significantly greater and appeared to show an age‐dependent effect. Mathematical modelling showed that the DIC‐EP formulation significantly increased diclofenac partitioning into the skin and a linear correlation was observed between steady‐state flux and the partition parameter. Greater skin deposition of diclofenac was also observed with DIC‐EP. These preliminary results suggest that the DIC‐EP formulation may be effective in treating inflammatory conditions in horses.     

Sperm cryopreservation in the Far‐Eastern wildcat (Prionailurus bengalensis euptilurus)

The Far‐Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far‐Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far‐Eastern wildcat captive‐born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen–thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far‐Eastern wildcat. The motility of frozen–thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far‐Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far‐Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far‐Eastern wildcat, respectively). Domestic cat epididymal and Far‐Eastern ejaculatory spermatozoa fertilized in vitro‐matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far‐Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.