Effect of semen thaw method on conception rate in four large commercial dairy heifer herds.

Semen processed with procedures intended to permit a flexible thaw method is used to breed millions of cows yearly. One method of thawing straws, the "pocket thaw" is used extensively with semen prepared with these procedures. Published field data is lacking for thaw method comparisons with semen processed to permit flexible-thawing. The objective of the present study was to measure the effect of semen thaw method (warm-water or pocket thaw) over all seasons and its interaction with herds, inseminators, straw package size, and sperm number on conception rate in commercial dairy heifer herds using semen processed with procedures historically optimized for success with flexible-thawing. Professional inseminators performed 11,215 services over a 16-month period in four large herds, achieving a 67.6% conception rate. Thaw method was alternated weekly. Thaw effect on conception status, determined by 70 days non-return rate, was estimated by a generalized linear mixed model. Neither thaw method nor number of sperm per straw significantly affected probability of conception (P=0.658 and 0.769, respectively). No interactions of thaw method with herd, sperm number, season, straw size, and straw size by season were detected (P=0.297, 0.526, 0.365, 0.723, and 0.824, respectively). Bull, herd, inseminator within herd, year, season, and straw size affected conception rate (P=0.002, 0.000, 0.000, 0.000, 0.000, and 0.014, respectively). In conclusion, for semen processed with procedures that permit flexible-thawing, thaw method (pocket thaw versus warm-water thaw) did not affect conception rate under commercial conditions and with routine semen handling methods.

Statistical methods for in situ hybridization: identification of autoradiographically labelled cells and structures.

In situ hybridization experiments frequently use autoradiography to identify labelled structures. Ideally, labelled cells will be overlain with a dense accumulation of particles, allowing one to discriminate them from unlabelled cells easily. However, if noise is high or the density of labelling is low, it can be difficult to distinguish bona fide labelling 'by eye'. In such situations, labelled cells could be overlooked. This paper evaluates two statistical solutions to this problem: (1) a parametric method proposed by Hashimoto and co-workers and (2) Wang & Wessendorf's non-parametric method using contingency testing (i.e. the chi-square or Fisher's exact tests). The Hashimoto method determines the mean and standard deviation of the density of background labelling, using sense-strand controls as the source of background levels. Cells labelled at densities greater than two standard deviations above the mean (P < 0.0455) are defined as significantly labelled. Contingency testing determines whether the grain density over a cell is significantly higher than that over the remainder of the image. When compared, the two methods gave similar results. The Hashimoto method may be more sensitive if most cells are labelled but contingency testing requires no assumptions about the uniformity of non-specific labelling.

Molecular cloning and tissue distribution of an avian D2 dopamine receptor mRNA from the domestic turkey (Maleagris gallopavo).

The reverse transcriptase-polymerase chain reaction (RT-PCR), in combination with 5' and 3' rapid amplification of cDNA ends (RACE), was used to clone a G protein-coupled receptor from turkey brain mRNA. This cDNA clone has an open reading frame of 1,311 base pairs encoding a 436-residue protein with seven transmembrane-spanning domains and exhibits high homology with previously cloned mammalian D2 dopamine receptors. Northern blot analysis of turkey brain mRNA detected an approximate 2.4-kb transcript. RT-PCR and subsequent nucleotide sequence analysis of turkey brain and peripheral tissue mRNA also demonstrated the presence of an alternatively spliced mRNA corresponding to the predicted D2 short isoform. RT-PCR experiments demonstrated a widespread distribution of alternatively spliced D2 dopamine receptor transcripts throughout the turkey brain and in select peripheral tissues as well. In situ hybridization experiments detected strong autoradiographic signals over much of the turkey telencephalon, diencephalon, mesencephalon, cerebellum, pituitary, and pineal gland. Dopamine has several important functions as a neurotransmitter and hormone in mammals and may have similar actions in avian species. The cloning and tissue distribution of the D2 receptor subtype should enable the investigation of any functional role dopamine and dopamine receptors exert on the physiology and behavior of birds.

D1 and D2 dopamine receptor messenger ribonucleic acid in brain and pituitary during the reproductive cycle of the turkey hen.

The regulation of prolactin secretion during the reproductive cycle of seasonal breeding birds appears to be largely under the stimulatory influence of hypothalamic vasoactive intestinal peptide (VIP). However, the factors influencing VIP secretion, and hence prolactin release, in birds remain largely unexplored. Recent evidence has demonstrated that dopamine and dopamine receptors may affect VIP and prolactin release in birds. The differential expression of dopamine receptors on hypothalamic VIP-releasing neurons may affect the degree of prolactinemia observed during the reproductive cycle of birds. In order to examine this hypothesis, we used reverse transcription-polymerase chain reaction to quantitate the levels of D1 and D2 dopamine receptor subtype mRNAs in the brain of the domestic turkey hen during the reproductive cycle. No significant difference in hypothalamic expression of D1 or D2 dopamine receptor subtypes during the reproductive cycle was observed. However, pronounced differences in D1D and D2 mRNAs were detected in cortex and cerebellum. Interestingly, there was a dramatic increase in pituitary D1D receptor mRNA during the reproductive stages of laying and incubation of eggs, which paralleled the hyperprolactinemic state of the turkey reproductive cycle. In addition, pituitary D2 receptor mRNA steadily increased throughout the reproductive cycle. In light of these observations, a modified hypothesis regarding the effects of dopamine on prolactin secretion is discussed.

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Bisbenzimide: a fluorescent counterstain for tissue autoradiography.

Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus, bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies.