Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

Standing Laparoscopic Peritoneal Flap Hernioplasty of the Vaginal Rings does not Modify the Sperm Production and Motility Characteristics in Intact Male Horses

Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this procedure based on 1‐year follow‐ups. There were no significant differences in the measured sperm variables (assessments based on the DSO, MOT, PMOT, VSL, VCL and VAP) during 1‐year follow‐ups.     

Changes in Bull Sperm Kinematics after Single Layer Centrifugation

The objective of this study was to evaluate bull sperm kinematics after centrifugation through a single layer of a colloid [Single Layer Centrifugation (SLC)]. Ejaculates from 20 bulls were extended and stored at 4–6°C for 24 h during transport to the laboratory for SLC through Androcoll‐B, followed by measurement of sperm kinematics in all samples. Total motility (86% and 88% for uncentrifuged and SLC samples, respectively) and progressive motility (84% for both the groups) were similar (p > 0.05). In contrast, straightness (STR) (0.65 vs 0.69), linearity (LIN) (0.32 vs 0.35) and beat cross frequency (BCF) (22.3 vs 23.6 Hz) were significantly higher in the SLC‐selected samples than in the uncentrifuged samples, whereas velocity of the average path (VAP) (95 vs 90 μm/s), curvilinear velocity (VCL) (192 vs 180 μm/s), amplitude of lateral head deviation (ALH) (7 μm vs 6.5 μm) and hypermotility (49% vs 38%) were significantly decreased. The kinematics of the samples with the poorest motility was improved most by SLC. In conclusion, even though SLC had no direct effect on total and progressive motility, it appeared to have a positive influence on several other kinematic parameters that may be important for fertilization after artificial insemination.     

Stimulation of bone formation in vitro and in rodents by statins

Osteoporosis and other diseases of bone loss are a major public health problem. Here it is shown that the statins, drugs widely used for lowering serum cholesterol, also enhance new bone formation in vitro and in rodents. This effect was associated with increased expression of the bone morphogenetic protein-2 (BMP-2) gene in bone cells. Lovastatin and simvastatin increased bone formation when injected subcutaneously over the calvaria of mice and increased cancellous bone volume when orally administered to rats. Thus, in appropriate doses, statins may have therapeutic applications for the treatment of osteoporosis.     

A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress

Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.     

Whooping crane recruitment enhanced by egg removal

To facilitate recovery through captive breeding and foster-parenting programs of the endangered whooping crane, one of two eggs was removed from 62% of nests in Wood Buffalo National Park during 1967-1996. Egg removals were justified because cranes usually rear a single chick; the other dies to siblicide or predation. Concerns exist that the wild population might have recovered even faster if nests had not been disturbed. Here we show, contrary to expectation, that removing one of two eggs from a whooping crane nest actually increases the probability of nest success, and this effect is highly correlated with the dynamics of predators in the nesting area. These results beg the question: why do whooping cranes lay two eggs? We attribute two-egg clutches to occasional “good years” in which both chicks survive, compensating for higher mortality in two-egg broods. Egg removal has benefited conservation allowing establishment of several captive flocks, supporting reintroduction of two new populations, and reducing the variance in reproductive success of the wild flock thereby minimizing extinction risk.     

Mortality of yellowtail flounder, Limanda ferruginea (Storer), eggs: effects of temperature and hormone-induced ovulation

Yellowtail flounder, Limanda ferruginea (Storer, 1839), is a highly marketable fish species that used to be a significant groundfish in the northwest Atlantic region. The potential of yellowtail flounder for mass rearing has recently been investigated, but studies into aspects of mortality during early ontogeny are lacking. We investigated the impact of spawning inducement on mortality of eggs from fertilisation until hatch, and the effects of rearing temperature on mortality and hatching time in this multiple batch spawning flounder. An ash-free dry mass (AFDM) technique for determining mortality was employed. This technique reduced rearing tank disturbances and provided quantitative mortality estimates. We found (1) that spontaneously ovulated (SO) mean egg mass (AFDM 24.84±0.73 μg) was significantly higher than mean egg mass from induced ovulation (IO) (AFDM 20.09±0.54 μg); (2) that mortality of SO eggs was lower than IO eggs (p=0.028), (3) that SO eggs reached a relatively constant (maximum) mortality by about day 3 and IO eggs by about day 5; (4) that there was no significant difference in mortality between eggs incubated at 10 °C and those incubated at 4 °C (p=0.320); and (5) that time to 100% hatch took significantly longer for eggs incubated at 4 °C (256.0±73.5 h) than those incubated at 10 °C (77.9±11.6 h) (p=0.003). There was considerable variation in egg mortality among batches and females; nevertheless, general trends indicate that GnRHa-induction of ovulation tends to decrease egg quality. In addition, the AFDM technique was an easily employed and accurate method of determining mortality. We recommend that unless coordination of spawning is important to a particular rearing protocol, induced ovulation should not be employed.     

Pasteurella multocida Heddleston serovars 1 and 14 express different lipopolysaccharide structures but share the same lipopolysaccharide biosynthesis outer core locus

Pasteurella multocida strains are classified using the Heddleston lipopolysaccharide (LPS) serotyping scheme into 16 serovars. Understanding the structural and genetic basis for this LPS typing scheme is important because protection against infections caused by P. multocida is generally considered to be serovar specific. Here we show that the serovar 14 type strain P2225 and the serovar 1 strains X73 and VP161 express similar LPS structures. However, the serovar 14 LPS lacks the terminal phosphocholine (PCho) residues present on the serovar 1 LPS and contains the 1,4-linked β-galactose but not the 1,6-linked β-galactose. Sequencing analysis of the LPS biosynthesis outer core loci of P2225 and the serovar 1 type strain X73 showed that they were nearly identical. However, the phosphocholine biosynthesis gene, pcgA of P2225 contained a 19bp nucleotide deletion. Complementation of P2225 with an intact pcgA resulted in an LPS structure identical to that expressed by serovar 1 strain VP161 and highly similar to that expressed by strain X73, with a 1,6-linked β-galactose and both terminal PCho residues. This study has shown unequivocally that strains belonging to serovar 1 and 14 share a common LPS outer core locus and that minor changes within this locus can dramatically alter the LPS structure expressed on the surface of P. multocida, and thus has implications into our understanding of the potential to generate cross-protective vaccines.     

Plasma indicators of muscle damage in a model of nutritional myopathy in weaner sheep

SUMMARY Subclinical nutritional myopathy was induced in 5-month-old sheep by feeding them a diet low in vitamin E and selenium. Subsequently clinical myopathy was induced by dosing with protected polyunsaturated fatty acids. Plasma activities of creatine kinase (CK), pyruvate kinase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase and aldolase, enzymes of muscle origin, all remained above their reference ranges in clinically affected sheep, but fluctuated widely. Similar fluctuations occurred in subclinically affected animals, resulting in some activities being within the reference ranges and some above, at different times. Plasma malondialdehyde, an indicator of lipid peroxidation, proved of no diagnostic value. Terminal plasma CK activities were significantly correlated with microscopic damage in the vastus lateralis (VL), but not the vastus intermedius (VI) or the tensor fascia lata (TFL) muscles. AST was the most highly correlated with damage in VI and VL. In two clinically affected sheep successfully treated with an oral dose of α-tocopherol actetate all enzymes decreased steadily to within their reference ranges, at rates probably related to their plasma half-lives. These results suggest that measurement of plasma CK activity would be useful in monitoring recovery of treated sheep.