Effects of pH, sodium bicarbonate, cryoprotectants and foetal bovine serum on the cryopreservation of European eel sperm.

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-cryopreservation.     

First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (‘cryolarvae’) were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.     

Changes in Testicular Interstitial Connective Tissue of Hamsters (Mesocricetus auratus) During Ageing and After Exposure to Short Photoperiod

The testicular interstitium of Syrian hamster (Mesocricetus auratus) was studied during ageing and in testicular regression after exposure to a short photoperiod, in relation to the interstitial cells and their connective tissue. This tissue was assessed histochemically using Masson's trichrome technique and the expression of Heat Shock Protein 47 (HSP‐47) and collagen IV (α5) was assessed in Leydig cells. Finally, an ultrastructural analysis of some cells of the testicular interstitium was made. Leydig cells were positive for HSP‐47 and collagen IV (α5). Ageing did not change the parameters studied while the short photoperiod altered the synthetic activity of Leydig cells. The positivity index of these cells for HSP‐47 was significantly higher in the regressed testis, but was lower for collagen IV (α5). During ageing no change were observed. Ultrastructural Leydig cells showed a discontinuous basal lamina that did not change during ageing. The basal lamina was not identified in Leydig cells regressed by exposure to a short photoperiod. In conclusion; the intertubular connective tissue suffers little change with age. By contrast, in the testis regressed after exposure to a short photoperiod the studied parameters related to the intertubular connective tissue were altered. These changes are probably related with the low synthetic activity of regressed Leydig cell.     

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years.

METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4–5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3–4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination.

RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISApositive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISAnegative.

CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (≥1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers.

CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.     

Administration of Thyroxine Affects the Morphometric Parameters and VEGF Expression in the Uterus and Placenta and the Uterine Vascularization but does Not Affect Reproductive Parameters in Gilts During Early Gestation

The aim of this study was to evaluate the effects of thyroxine administration on morphometric parameters, expression of vascular endothelial growth factor (VEGF) and vascularization in the uterus and placenta and reproductive parameters in gilts at 70 days of gestation. At 150 days of age, i.e., before first heat, 20 gilts were randomly divided into two experimental groups: treated (n = 10) and control (n = 10). The treated group received a daily dose of 400 μg of l ‐thyroxine (T₄) in their diet until slaughter and the control group received only typical meals. Before artificial insemination, blood was collected to determine plasma total T₄. The gilts were inseminated in the second oestrus and slaughtered at 70 days of gestation. The foetal thyroid follicular epithelium height, number, size and weight of foetuses; foetal myogenesis, corpora lutea number, embryonic mortality rate, uterine weight, placental weight and placental fluid volume were measured. Histomorphometric and immunohistochemical analysis of uterus and placenta were determined. The averages of all variables were compared by the Student’s t‐test. The gilts treated with thyroxine showed significant increase of plasma total T₄. At 70 days of gestation, the heights of the trophoblastic epithelium, endometrial epithelium and endometrial gland epithelium were significantly higher in the group treated with T₄. The expression of cytoplasmatic and nuclear VEGF in trophoblastic cells and the number of blood vessels per field in endometrial stroma were significantly higher in the gilts treated with T₄. No other significant differences between groups were obtained with respect to other parameters (p > 0.05). We conclude that oral administration of T₄ up to 70 days of pregnancy in gilts affects the morphometric parameters, the expression of placental VEGF and the uterine vascularization but does not affect reproductive parameters in gilts during early gestation.     

Polyolefin spheres from metallocenes supported on noninteracting polystyrene

To obviate the destructive interaction of highly reactive metallocene catalysts with classical silica-based supports while retaining the advantage of supported catalysts, a noninteracting polystyrene support was developed. Supported catalysts for the polymerization of alpha-olefins are prepared by treating lightly cross-linked, chloromethylated polystyrene beads consecutively with a secondary amine, an ammonium salt of a weakly coordinating anion, and a neutral dialkylmetallocene. Catalytic sites are distributed homogeneously throughout the support particle, and the polymerization occurs within the bead, in contrast to traditional surface-supported metallocene catalysts. The copolymerization of ethylene and 1-hexene at 40 degreesC affords discrete spherical polyolefin beads with a size (0.3 to 1.4 millimeters) that varies according to the polymerization time.     

Spina bifida with associated malformations of the central nervous system in Dorper-cross sheep

Abstract

CASE HISTORY: In 2008, six lambs within a flock of Dorpercross sheep were born with musculoskeletal and neurological disease. Clinical signs included hindlimb weakness, and urinary incontinence.

CLINICAL FINDINGS: All lambs had focal, inverted areas of alopecic skin over the caudal sacrum, and short, often kinked tails. Four affected lambs were subject to euthanasia, and necropsied. On gross examination, the arches of sacral vertebrae were absent, and spinal nerves and meninges were adherent to the overlying subcutis. Other gross lesions included narrow, elongated skulls, herniation of the occipital lobes into the caudal fossas, hydrocephalus, and syringomyelia. One lamb had coning of the cerebellar vermis, but cerebellar herniation through the foramen magnum was not identified.

DIAGNOSIS: Spina bifida, with associated malformations of the central nervous system.

CLINICAL RELEVANCE: Examination of breeding records suggested either an autosomal recessive or partially penetrant autosomal dominant pattern of inheritance. Because of the associated tail lesions it is proposed that the pathogenesis of this syndrome involves a defect in development of the tail bud (secondary neurulation), that tethering of the spinal cord resulted in the clinical signs, and abnormal pressure of the cerebral spinal fluid resulted in the defects in the skull and brain.     

Facial swelling and discharging lesions associated with abnormalities of the mandible in kunekune pigs

Abstract

CASE HISTORIES: Four adult kunekune pigs developed facial swelling at the base of the right ear that ruptured and discharged food material. A further six pigs that had similar clinical signs were reported by members of the New Zealand Kunekune Association who responded to an email survey, one of which was confirmed by post-mortem examination.

CLINICAL FINDINGS: Inside the mouth of each pig there was an opening at the junction of the body and ramus of the mandible just lateral to the most caudal visible molar that was impacted with masticated feed. The food packed into the mandible resulted in infection and progressive erosion of the medullary cavity of the bone until it reached the ramus where it eroded through the lateral cortex. The feed material then tracked through the soft tissues to form a subcutaneous abscess, which eventually ruptured resulting in a draining lesion. In Case 2, which had had the lesion for 2 years, the cavity in the mandible was lined with mucosa that had healed to the skin to produce a fistula. In all four pigs there was also a lesion in the left side of the mandible that was not as developed as that on the right side.

DIAGNOSIS: The facial swellings were produced by feed material that had impacted into the mandible through an opening immediately caudal to the cheek teeth and then emerged through one or more lesions in the lateral aspect of the ramus of the mandible.

CLINICAL RELEVANCE: Although it has not been previously reported, anecdotal reports and our survey suggest that this condition may occur relatively frequently in kunekune pigs. It should be considered as a differential diagnosis for facial swellings and discharging lesions in these animals.