Effect of Semen Extender and Density Gradient Centrifugation on the Motility and Fertility of Turkey Spermatozoa

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Functional Activity of Frozen Thawed Chinchilla lanigera Spermatozoa Cryopreserved with Glycerol or Ethylene Glycol

The cryopreservation of spermatozoa constitutes a valuable tool for the captive breeding management of valuable and/or threatened species. Chinchilla lanigera is a species almost extinct in the wild, and the domestic counterpart has one of the most valuable pelts in the world. The objectives of this study were to: (i) compare the functional activity of post‐thawed chinchilla spermatozoa cryopreserved at ?196°C either with glycerol (G) or ethylene glycol (EG) as cryoprotectants (1 m final concentration) and (ii) investigate the effects of incubating the gametes for 4 h in the presence or in the absence of the cryoprotectants; evaluations were performed taking into account motility, viability, response to hypo‐osmotic shock and acrosome integrity of the cells. Parameters reflecting post‐thaw (0 h) sperm functional activity were significantly lower than those of freshly ejaculated gametes. When comparing the cryoprotectant efficiency of G vs EG, neither cryoprotectant agent offered appreciable advantages. After 4 h of incubation, in the presence or absence of the cryoprotectant agent, a rapid and significant decrease was found in all functional parameters and remained at ～ 20–30% motile, viable and viable acrosome intact cells. Viability was significantly lower when the cryoprotectant was removed from the media (possibly due to the centrifugation process). With respect to the maintenance of sperm membrane integrity, only ～ 10% of cells showed membrane resistance to hypo‐osmotic conditions after the 4 h incubation period. These results constitute new insights for cryopreservation protocols and the development of assisted reproductive techniques in this species.     

Extent-dependent habitat selection in a migratory large herbivore: road avoidance across scales

Context

In southwestern Alberta, human development, including roads, is encroaching on the landscape and into the range of a partially migratory population of elk (Cervus elaphus).

Objectives

To quantify factors influencing among- and within-home-range selection of winter range in this population.

Methods

We studied individual habitat selection and road avoidance at two biologically relevant spatial scales. We outlined availability extents for 107 individual elk-years based on observed fall migration distance, and based on a minimum convex polygon around winter telemetry relocations. To model the response by elk to road disturbance, we fit a habitat-selection model to each elk-year at each of the two availability extents, and examined population-level and individual variation in space-use. We then evaluated the relationship between inferred selection at the two scales and the functional response in selection.

Results

Roads had a ubiquitous influence on elk across scales. Elk, individually and as a population, avoided roads when migrating to their winter range and within this seasonal home range. Individual elk that avoided roads more strongly relative to the population did so at both scales of analysis.Further, the avoidance of low-use roads decreased with increasing road density. These results support bottom-up habitat-selection patterns (i.e., scale-independent) and functional response in habitat selection.

Conclusions

Overall, using a multi-scale habitat selection analysis, we show that road avoidance is a major determinant of elk space-use behaviour across multiple scales. Consequently, any new road construction or increases in road-use intensity could have detrimental effects on migratory elk populations by restricting space-use.

     

Food ration requirements of 0 yellowtail flounder Limanda ferruginea (Storer) juveniles

Several studies have shown that food ration can affect the growth of cultured fish. Determining the optimal food ration would help to achieve better growth and also provide direct economic benefits due to reduced food wastage, which would lead to commercial success. Therefore, we studied the effects of ration levels on growth performance of 0⁺ juvenile yellowtail flounder to determine the optimal food ration. Two experiments were conducted; the first experiment as a preliminary using ration levels of 1%, 2%, 4%, 6% body weight per day (% bw day⁻¹) held at 7.0 °C with a stocking density of 0.95 kg m⁻² (45% bottom coverage). Results of this preliminary experiment indicated that fish fed with 1% bw day⁻¹ had significantly lower growth (weight, length, body depth and specific growth rates (SGR)) than those fed with 2%, 4% and 6% ration. However, fish fed with rations of 1% and 2% showed significantly lower gross food conversion ratios (GFCR) than fish fed with 4% and 6% rations. Survival was not significantly affected by different ration levels. Based on these preliminary results, we used ration levels of 1%, 1.5%, 2% and 3% for the main experiment. Fish were held at 10 °C with a stocking density of 1.45 kg m⁻² (34% bottom coverage). Results indicated that fish fed with 1%, 1.5% and 2% bw day⁻¹ had significantly lower growth than fish fed with 3% bw day⁻¹. GFCR was significantly different for all four rations. It was lower for 1% than 1.5%, 2% and 3% rations. Survival was not significantly different between any treatments. We discuss our results with emphasis on growth and economics (i.e., feed wastage) and stress the need to balance both components in a commercial operation.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Salmonella Enteritidis: the egg and I

SUMMARY: The world-wide clinical incidence of Salmonella Enteritidis has increased markedly. The increase is associated with the enhanced ability of the bacterium to systemically colonise layer chickens. Subsequent contamination and consumption of intact shell eggs from colonised layer hens, either directly or in foods containing raw or lightly cooked eggs, causes human disease. Despite investigation, no change in the biology of the bacterium has been correlated with increased colonisation in chickens. To date, no method of control at the production level has proven effective; consumer education is the best means of minimising the public health risk.     

Fertilisation and weed control effects on yield and weeds in durum wheat grown under rain-fed conditions in a Mediterranean climate

Summary A field study was undertaken to examine the effect of fertiliser type and weed control system on grain yield, yield components and weed population in durum wheat ( Triticum turgidum. var. durum) grown in a 4-year rotation [bare fallow–barley ( Hordeum vulgare )–vetch ( Vicia sativa )–wheat]. Fertilisation treatments were: no fertilisation, organic fertilisation with 2500 kg ha⁻¹ of compost (sheep manure and cereal straw) and chemical fertilisation with 100–60–60 kg ha⁻¹ of NPK. Weed-control treatments were: no control, herbicide, harrowing with long-flex spring tines and strip sowing with inter-row hoeing. Chemical fertilisation increased grain yield with respect to the other treatments, amongst which no significant differences were noted. Weed-control systems afforded no improvement in yield compared with controls; indeed, yields obtained using inter-row hoeing were lower. The weed population consisted of a large variety of species, of which the most important were Convolvulus arvensis and Polygonum aviculare . Application of weed-control systems reduced weed density. Herbicide was the most effective, reducing weed biomass by 80%, whilst harrowing and hoeing reduced weed biomass by 40% and 52% respectively. Inter-row hoeing does not appear to be a viable alternative to herbicides, when used as the sole weed control method in a non-diverse cropping system in Mediterranean climates. The lack of response to compost suggests a need for further long-term research.     

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.