Inmaculada Rodríguez

Gestation length in Carthusian Spanishbred mares

The length of gestation in Carthusian Spanishbred mares reared in southern Spain was calculated on the basis of 364 spontaneous full-term deliveries taking place in the 7-year period 1994–2000. Ultrasonography was used to determine follicular dehiscence (1st day of gestation) and to confirm conception in mares. Mean gestation length was 338.95±9.55 (SD) days, and a normal range of 319–359 days was established for this breed. Pregnancy with a female foal was 2.17 days shorter than with a male foal (P<0.05). Gestation length shortened as the breeding season progressed: for each month later in the breeding season that a mare was mated there was an decrease in gestation length of 2.75 days. The variation in gestation length was greater when mating took place during the reproductive season.

Assessment of goat semen freezability according to the spermatozoa characteristics from fresh and frozen samples

A total of 110 ejaculates were assessed in order to determine the influence of the physical parameters of goat semen on post-thaw motility and acrosome integrity. Sperm ejaculate characteristics, sperm motility, morphology and acrosome integrity were assessed in fresh and frozen samples by the sperm class analyzer (SCA) and Spermac staining technique. A decrease in acrosome integrity and sperm motility was found after thawing (P<0.01). Six semen parameters assessed before freezing were identified as predictors of sperm freezability (P<0.01). The percentage of morphological abnormalities (R=0.856) and motile sperm cells (R=0.655) in fresh semen are the best predictors to know the total post-thaw variability parameters.

Use of ultrafast Papanicolaou stain for exfoliative vaginal cytology in bitches

EXFOLIATIVE vaginal cytology has been established as a useful clinical tool for studying the oestrous cycle phase in bitches in order to optimise the timing of breeding (Tammer and others 1994), although some investigators disagree with this suggestion (Hiemstra and others 2001). The technique has also been used as a complementary tool in the diagnosis of certain infectious or inflammatory pathologies and tumours (Roszel 1975, Wright and Parry 1989, Johnson 1991). Romanowsky-type staining procedures are widely used in canine exfoliative vaginal cytology because of their versatility and ease of use. These are based on the visualisation of the nucleus and on differences in cell morphology. However, commercially developed polychrome stains have improved cytological diagnosis by revealing more complete information about the specimen under study (Mialot 1984). The Papanicolaou stain (Papanicolaou 1942) has for many years been the most widely used polychrome technique in human vaginal cytology; however, the highly complex and timeconsuming nature of this procedure led to the development of a modification known as the ultrafast Papanicolaou protocol, which was initially designed to stain fine-needle aspiration biopsies for diagnosis of a range of human tumours (such as carcinoma, adenocarcinoma, schwannoma and breast lesions) (Yang 1995, Yang and Alvarez 1995). This procedure enables processing times to be reduced to five minutes, and provides clear polychromatic images, free of erythrocytes, and with a slight increase in cell size. The procedure is basically the same as the traditional Papanicolaou stain protocol, except that the duration of each step is shortened. Haematoxylin is responsible for nuclear staining, and orange G and EA50 give orange/pink and blue coloration, respectively, to cytoplasm. This rapid polychromatic stain offers more information than monochromatic stains, since a blue to orange colour transition can be observed in the cytoplasm. The cytoplasm in vaginal epithelial cells is often stained blue, but in the luminal surface cells, due to the presence of intracellular keratohyaline, this coloration changes to orange. This precursor of keratin appears as an effect of the predominance of plasmatic oestrogens, which induce proliferation, maturation and keratinisation in the vaginal epithelium. This short communication describes the use of the ultrafast Papanicolaou stain in canine exfoliative vaginal cytology. Vaginal specimens were taken from 30 bitches examined at the Veterinary Hospital of the University of Cordoba, in order to determine accurately the optimal time for natural or artificial insemination. Vaginal smears were prepared daily until the oestrous cycle was complete. When a serosanguineous vaginal discharge was abundant during the oestrus cycle, the area was cleaned before specimen collection. The labia were separated with one hand and a saline-dampened, sterile, cotton swab was introduced into the dorsal aspect of the labial commissure with the other. The swab was inserted dorsocranially as far as the dorsoanterior vaginal portion and then withdrawn; it was then rolled on to a clean, prelabelled slide, which was air dried. The smears were stained following the ultrafast Papanicolaou protocol (Yang and Alvarez 1995) (Table 1). All the smears were interpreted by the same technician in order to avoid subjective errors. The eosinophilic index (the number of vaginal epithelial cells with orange cytoplasm divided by the total number of cells, expressed as a percentage) was used to identify keratinised cells (Badinand and Fontbonne 1993). In addition, the plasma progesterone concentration was measured (Ovucheck Premate; Vetoquinol) at the same time, as an indicator of ovulation and as a reliable hormonal indicator of the optimal time of breeding. The determination of the optimal breeding time was accurate in all the bitches based on the proportion of cellular types. The animals did not present any medical or laboratory evidence of hormonal anomalies during oestrous cycles. However, when hormonal anomalies should have arisen, diagnosis by polychrome strains would have been the most suitable. Exfoliative vaginal cytology in bitches is based on the evaluation of changes in the morphological and staining characteristics of cells throughout the oestrous cycle, which occur as a consequence of hormonal influences. The vaginal epithelial cells are classified, from the deepest to the most superficial layer, as parabasal, small and large intermediate, superficial and squamous types. Parabasal is the smallest cell normally seen; these cells have a round morphology, the nucleus is large, containing compacted chromatin, and the cytoplasm is basophilic. Intermediate cells are larger and rounded, and are classified as small or large types; the nuclear:cytoplasmic ratio is the lowest of the vaginal epithelial cell types, and basophilic cytoplasm is usually present. Superficial cells are situated in a more superficial layer and appear flattened and partially cornified, with a pyknotic (small and dark) nucleus, and the abundant acidophilic cellular cytoplasm stains orange. Squamous cells are the most differentiated cells in the vaginal epithelium; they are the most superficial cells, have angular margins and lack nuclei. These fully cornified cells show acidophilic (orange) staining. Cell type variations, in addition to changes in behaviour and in the external genitalia, are characteristic of some phases of the oestrous cycle. The anoestrus stage in the bitch, which lasts for approximately four months, is known as the sexual rest period, in which the levels of sexual hormones are basal. During this phase, the vaginal epithelium is formed of parabasal and intermediate cells, and stains with a basophilic (blue) coloration. The next phase is pro-oestrus, which lasts Veterinary Record (2005) 156, 648-650

Viability of honeybee colonies exposed to sunflowers grown from seeds treated with the neonicotinoids thiamethoxam and clothianidin

In this study, honeybee colonies were monitored in a field study conducted on sunflowers grown from seeds treated with the systemic neonicotinoids thiamethoxam or clothianidin. This field trial was carried out in different representative growing areas in Spain over a beekeeping season. The health and development of the colonies was assessed by measuring factors that have a significant influence on their strength and overwintering ability. The parameters assessed were: colony strength (adult bees), brood development, amount of pollen and honey stores and presence and status of the queen. The concentration of residues (clothianidin and thiamethoxam) in samples of beebread and in adult bees was at the level of ng.g-1; in the ranges of 0.10-2.89 ng g-1 and 0.05-0.12 ng g-1; 0.10-0.37 ng g-1 and 0.01-0.05 ng g-1, respectively. Multivariate models were applied to evaluate the interaction among factors. No significant differences were found between the honeybee colonies of the different treatment groups, either exposed or not to the neonicotinoids. The seasonal development of the colonies was affected by the environmental conditions which, together with the initial strength of the bee colonies and the characteristics of the plots, had a significant effect on the different variables studied.