Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.     

Identification of tissue hypoxia in the livers of ascitic and hypoxia‐induced broilers using trypan blue

1. The perfusion of livers with the vital dye trypan blue was performed to test for evidence of tissue hypoxia in 3 groups of young broiler chickens, namely, ascitic, hypoxia‐induced and controls.

2. Hepatocytes that stained with trypan blue were considered to be dead or dying before fixation and represented damaged cells.

3. The proportion of trypan blue‐stained hepatocytes in the livers of ascitic birds was slightly less than half that observed in the hypoxia‐induced birds but significandy more than the proportion of stained cells observed in control birds.

4. Liver damage in the ascitic birds was also assessed biochemically by an altered enzyme profile.

5. The study demonstrated that increased trypan blue uptake in the livers of ascitic birds reared at sea‐level may be the consequence of hypoxia stress caused by reduced oxygen utilisation.     

Effects of dietary lysine and energy density on performance and carcass characteristics of finishing pigs fed ractopamine

Two hundred sixteen crossbred barrows and gilts (84.3 kg BW) were used to test the effects of dietary energy density and lysine:energy ratio (Lys:ME) on the performance, carcass characteristics, and pork quality of finishing pigs fed 10 ppm ractopamine. Pigs were blocked by BW and gender, allotted to 36 pens (six pigs per pen), and pens were assigned randomly within blocks to dietary treatments (as-fed basis) arranged in a 2 x 3 factorial design, with two levels of energy (3.30 or 3.48 Mcal/kg) and three Lys:ME (1.7, 2.4, or 3.1 g lysine/Mcal) levels. Pigs were fed experimental diets for 28 d, and weights and feed disappearance were recorded weekly to calculate ADG, ADFI, and G:F. Upon completion of the feeding trial, pigs were slaughtered and carcass data were collected before fabrication. During carcass fabrication, hams were analyzed for lean composition using a ham electrical conductivity (TOBEC) unit, and loins were collected, vacuum-packaged, and boxed for pork quality data collection. Energy density had no (P > 0.22) effect on ADG or ADFI across the entire 28-d feeding trial; however, pigs fed 3.48 Mcal of ME were more (P < 0.02) efficient than pigs fed 3.30 Mcal of ME. In addition, ADG and G:F increased linearly (P < 0.01) as Lys:ME increased from 1.7 to 3.1 g/Mcal. Carcasses of pigs fed 3.48 Mcal of ME were fatter at the last lumbar vertebrae (P < 0.08) and 10th rib (P < 0.04), resulting in a lower (P < 0.03) predicted fat-free lean yield (FFLY). Conversely, 10th-rib fat thickness decreased linearly (P = 0.02), and LM depth (P < 0.01) and area (P < 0.01) increased linearly, with increasing Lys:ME. Moreover, FFLY (P < 0.01) and actual ham lean yield (P < 0.01) increased as Lys:ME increased in the diet. Dietary energy density had no (P > 0.19) effect on pork quality, and Lys:ME did not (P > 0.20) affect muscle pH, drip loss, color, and firmness scores. Marbling scores, as well as LM lipid content, decreased linearly (P < 0.01) as Lys:ME increased from 1.7 to 3.1 g/Mcal. There was a linear (P < 0.01) increase in shear force of cooked LM chops as Lys:ME increased in the finishing diet. Results indicate that 3.30 Mcal of ME/kg (as-fed basis) is sufficient for optimal performance and carcass leanness in pigs fed ractopamine. The Lys:ME for optimal performance and carcass composition seems higher than that currently used in the swine industry; however, feeding very high Lys:ME (> 3.0 g/Mcal, as-fed basis) to ractopamine-fed pigs may result in decreased marbling and cooked pork tenderness.     

Book reviews

Amino Acids in Farm Animal Nutrition. Edited by J. P. F. D'Mello. 1994. 417 pp. £55.00 Wallingford, Oxon, CAB International. ISBN 0 85198 8814

Avian Hematology and Cytology. Terry W. Campbell. 1995. 104 pp. Illustrated. £48.00. Ames, Iowa State University Press. ISBN 0 8138 2970 4.     

Terraclor (PCNB) for control ofRhizoctonia solani. II. PCNB residues in tubers

Terraclor (PCNB) applied to soils by broadcast and row slightly reduced the incidence ofRhizoctonia solani but did not increase yield or per cent of U. S. No. 1 tubers either by weight or numbers. Residues of PCNB in tubers grown in treated soils increased as the rate per acre of PCNB increased. Almost all of the residues were in the tuber peel.     

Welfare assessment of broiler breeders that are food restricted after peak rate of lay

1. Broiler breeder females were reared on one of three growth curves (ad libitum, conventional or modified restriction) and given rations containing a high or low concentration of crude protein. After the peak rate of lay they were fed ad libitum or a decreasing quantity of food in response to declining egg production. The welfare of the birds was assessed by determining the changes in indices of welfare at 36, 48 and 60 weeks of age. 2. Body weight increased rapidly in restricted birds fed ad libitum post-peak and water intake declined. 3. Post-peak food restriction was associated with a decrease in resting and increased drinking and spot-pecking activities. Birds that were food restricted during rearing spent more time foraging and spot-pecking at 36 and 48 but not 60 weeks of age. 4. Immune function increased with age but was not affected by the experimental treatments. The heterophil-lymphocyte ratio in birds fed ad libitum during rearing was numerically lower at 36 and higher at 48 and 60 weeks of age compared with restricted birds. 5. There was no effect of treatment on plasma corticosterone concentration. Creatine kinase activity was high at 60 weeks in treatments that were characterised by poor reproductive status and the activities of other enzymes reflected differences in reproductive status and mortality. 6. There was no long-term welfare or production advantage from feeding low protein rations or more generous feeding during the rearing period compared with conventional food restriction programmes.     

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.     

Production and oxidation of wool grease after shearing

OBJECTIVE: To measure the production and amount of oxidation of wool grease secreted immediately after shearing. To identify components of wool grease that might act as a carrier to facilitate lateral diffusion of topically applied insecticides. DESIGN: Fine-wool Merino sheep were shorn and residual greasy wool was collected from the sheep's flank. The quantity of grease produced, and the amount of oxidation was measured during 18 days after shearing. Wool grease was fractionated into five component groups based on their polarity and the degree of oxidation in these fractions determined. RESULTS: There was a 24% increase in grease production within 2 days after shearing but secretions returned to pre-shearing amounts after 4 days. During this period wool grease oxidized rapidly. Of the grease fractions examined, sterol and wax esters remained essentially unoxidized whereas free sterols such as cholesterol and lanosterol, fatty acids and polar lipids, aldehydes and alcohols were extensively oxidized within 7 days after shearing. CONCLUSION: The transient increase in grease production after shearing may facilitate diffusion of topically applied synthetic pyrethroid insecticides. Oxidation of grease components may then contain the insecticide and limit further diffusion. Incorporating the insecticide in non-oxidising fractions of wool grease may make insecticide dispersion more efficient.