Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.

One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen production, it is likely that more than one method for cryopreserving sperm will be necessary. This manuscript reviews some of the processes involved in cryopreservation, how individual sperm physiology affects the ability to survive freezing and thawing, and how cryopreservation protocols can be customized to maximize sperm cryosurvival on an individual stallion basis.

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli.

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steadystate length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells.

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility.

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

The equine frozen semen industry.

Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major impediments to the development of the frozen semen industry include 1. Lower fertility with frozen semen as compared to cooled semen for many stallions. 2. Increased costs associated with management of mares for AI with frozen semen using current insemination protocols. 3. Unfavorable marketing practices for frozen semen. Reports of fertility with cooled transported semen in commercial breeding programs indicate seasonal pregnancy rates ranging from 60 to 90%. We compiled data from three commercial transported cooled semen programs in which semen from 16 stallions was used for insemination of 850 mares throughout North America by local veterinarians. During the 1999 and 2000 breeding seasons, first cycle and seasonal pregnancy rates of 59.4 and 74.7% were obtained. During that same period, first cycle and seasonal pregnancy rates of 51.3 and 75.6% were obtained following insemination of 876 mares with frozen semen from 106 different stallions processed by our laboratory and distributed through our commercial distribution program. First cycle and seasonal pregnancy rates were higher for mares bred outside of North America than for mares bred within North America (53.5 and 81.9 versus 49.4 and 65.6%, respectively). Seasonal pregnancy rates were higher presumably because of the better mare management employed for mares bred with exported semen and the fact that some of the domestic mares were switched to cooled semen insemination after a failed first cycle attempt with frozen semen. These data support the position that comparable seasonal pregnancy rates may be obtained using frozen and liquid cooled semen in a commercial setting.

Fatal cervical necrotizing fasciitis (a report of two cases of confirmed odontogenic origin and one of possible odontogenic origin).

Three cases of cervical necrotizing fasciitis (CNF), two of confirmed odontogenic origin and one of probable odontogenic origin, were observed from 1993-1999. This is in addition to three cases previously reported by this office. A rare sequelae of dental infection, CNF can be a severe, rapidly progressing infection of the cervical tissues having a mortality rate of up to 50%. "Hospital gangrene" was first described during the Civil War. It was later to be described as necrotizing fasciitis and later yet was designated as a separate clinicopathological diagnosis.

Alterations in neural intermediate filament organization: functional implications and the induction of pathological changes related to motor neuron disease.

The properties regulating the supramolecular organization of neural intermediate filament (NIF) networks have been investigated in cultured dorsal root ganglion (DRG) neurons. The studies described take advantage of the ability of endogenous NIF to incorporate purified biotinylated neurofilament triplet (NFT) proteins, NF-L, NF-M and NF-H. When injected at concentrations of 0.8-1.0 mg/ml injection buffer, each of these proteins is incorporated without perturbing the endogenous NIF network. However, at progressively higher concentrations, NF-H induces the aggregation and accumulation of NIF in the cell body. Subsequent to the induction of these aggregates, numerous alterations in the cytoarchitecture of neurons can be detected. The latter occur in a temporal sequence which appears to begin with the fragmentation of the Golgi complex. At later times, accumulation of mitochondria within the proximal region of neurites, peripheralization of the nucleus, and a significant decrease in neurite caliber become obvious. After longer time periods, the NIF aggregates are seen to react with an antibody which reveals abnormally phosphorylated NF-H. These observations demonstrate that an imbalance in the normal stoichiometric relationships among the NFT proteins rapidly alters the supramolecular organization of the NIF network. These changes most likely reflect the normal functions of neurofilaments in cell shape and the organization and cytoplasmic distribution of membranous organelles. Interestingly, virtually all of these changes closely resemble those which have been reported in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). These findings suggest that cultured neurons can be used as models for more precisely defining the relationships between the formation of NIF aggregates and the sequence of cytopathological events which typify neurodegenerative diseases.

Solution and solid-state circular dichroism analyses of a human salivary proline-rich glycoprotein repeating domain and its subfragments.

Solution- and solid-state c.d. spectra, as well as surface energetics values, were collected for a series of peptides derived from human salivary proline-rich glycoprotein (PRG). The acronyms and sequences for these peptides are as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)-P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2, and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. The presence of stable poly-L-proline II-like 'mini' helices in the solution state was found to be dependent on peptide chain length, pH, salt, and organic solvent type. Other conformational features such as kinks and beta-/gamma-turns were also found in the larger peptides. Solid-state peptide conformations were not necessarily related to their solution-state counterparts. Poly-L-proline II-like 'mini' helices, kinks, and beta-/gamma-turns were similarly found in the various substrate-bound PRG9 peptides. Surface energetics parameters suggested specific orientations for PRG9 peptides and their constituent acids and homopolymers.

A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau.

We previously reported the presence of the microtubule-associated protein, tau in the nuclei of primate cells in culture. The present study confirms the existence of nuclear tau in two human neuroblastoma cells lines by indirect immunofluorescence and Western blot using mAbs to tau. Northern blot analysis of poly A+ mRNA detects a novel 2-kb tau transcript coexpressed with the 6-kb message in cultured human cells and human frontal cortex. PCR and cDNA sequencing demonstrate that the 2-kb message contains the entire tau coding region. Furthermore, actinomycin D transcription inhibition experiments indicate that the 2-kb message is not derived from the 6-kb message, but instead arises from the original tau transcript. One of the human neuroblastoma cell lines examined contains both nuclear and cytoplasmic tau as assayed by both Western blot and indirect immunofluorescence. Northern blot analysis of this cell line indicates that copious amounts of the 2-kb message are present while little of the 6-kb transcript is obvious. Immunofluorescence analysis of this cell line demonstrates that the cytoplasmic tau is not localized to microtubules. Together, these results indicate that the 2-kb tau message in humans may specify tau for non-microtubule functions in both the cytoplasm and the nucleus. We hypothesize that this is accomplished via a message targeting mechanism mediated by the untranslated regions of the tau messages.

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.

Identification of nuclear tau isoforms in human neuroblastoma cells.

The tau proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, tau has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer disease and of most aged individuals with Down syndrome (trisomy 21). We report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, we further demonstrate the existence of the entire tau molecule in the isolated nuclei of neuroblastoma cells. Nuclear tau proteins, like the tau proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that tau may function in processes not directly associated with microtubules and that highly insoluble complexes of tau may also play a role in normal cellular physiology.

Fertility of unfrozen and frozen stallion spermatozoa extended in EDTA-lactose-egg yolk and packaged in straws.

The fertility of frozen-thawed semen was compared with that obtained using fresh semen extended in skim milk. Semen for freezing was obtained in June from four stallions of unknown fertility; two ejaculates were collected 1 to 2 h apart every 3 or 4 d. The gel-free fraction of the ejaculate was mixed 1:1 with a glucose-EDTA solution (disodium ethylenediaminetetraacetic acid) and centrifuged at 650 x g for 15 min. The spermatozoa were resuspended in an EDTA-lactose-egg yolk extender containing 5% glycerol. Semen was frozen in .5-ml French straws containing 250 x 10(6) progressively motile spermatozoa before freezing. Only for 31% of the 54 ejaculates frozen was post-thaw spermatozoal motility greater than or equal to 50% of the percentage of progressively motile spermatozoa observed during evaluation of the neat semen. Spermatozoa in second ejaculates apparently were more susceptible to the adverse effects of dilution and centrifugation than spermatozoa in first ejaculates. Only samples containing greater than 30% progressively motile spermatozoa after freezing and thawing (at 38 C) were used for insemination. In June and July, 101 mares were inseminated daily with semen from one of three stallions beginning on d 2 and continuing through the end of estrus for one cycle. Mares were inseminated with semen in one straw or with 250 x 10(6) progressively motile spermatozoa extended in 10 ml of skim milk. Because of the poor survival of spermatozoa that had been frozen and thawed, mares inseminated with frozen-thawed semen received 100 to 130 x 10(6) progressively motile spermatozoa as compared with 250 x 10(6) progressively motile spermatozoa for mares inseminated with fresh semen. One cycle pregnancy rates, based on rectal palpation 50 to 60 d after ovulation, were 29% using frozen-thawed semen and 66% using fresh semen (P less than .05). Values for individual stallions were 19, 24 and 47% with frozen semen and 47, 61 and 67% with fresh semen. Routine use of frozen stallion semen is not recommended at this time.