In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains from two hybrids, WF9xH55 (W) and K64xK55 (K) were collected and a sample from each was cultured immediately (0 h). The remainder was subdivided and stored at 2, 20, and 35° C. At 3, 6, 12, 24, 36, 48, 72, 96, and 120 h, a sample was cultured. The culture medium contained 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid. Storage at 2° C resulted in a large increase in germination percentage in both W and K reaching a maximum at 24 h and then slowly decreasing with additional storage. No germination was observed at 96 h with W and at 120 h with K. The complete loss of germination occurred during a 24 h period and was very abrupt. At 20° C, a similar but less pronounced pattern was observed. However, after 24 h, aggregates of 50–1 000 pollen grains developed during storage in both W and K. Storage of W at 35° C slightly decreased the germination percentage at 3 h and eliminated it at 6 h. Storage of K at 35° C substantially increased the germination percentage at 3 h with further increases in storage periods resulting in the aggregation of grains. This general pattern of an increase at shorter storage periods followed by a gradual decrease as the storage period was extended, was found for pollen tube length and growth rate. In vitro germination characteristics can be substantially altered by the temperature and length of storage and the response to storage is associated with pollen source.Journal Series Paper No. 4566, Florida Agricultural Experiment Station.     

Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender

This study tested whether variable temperatures (from −0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at −0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 °C, 12 °C to 15 °C, respectively). Per cycle pregnancy rate (PC) was measured after one artificial insemination (AI) in uterine body of 200 × 10⁶ total spermatozoa, 7 h (Experiment 1) or 17 h (Experiment 2) before ovulation. In Experiment 1, PC was similar for both conditions (60% (n = 40 cycles) vs. 63% (n = 40), respectively, 5 stallions × 8 cycles). Only velocity VCL and ALH were slightly higher at 15 °C. In Experiment 2, PC was reduced when ambient temperature was low (semen at −0.5 °C to 3 °C; PC = 25%) rather than intermediate (semen at 4 °C to 7 °C; PC = 53%) or high (semen at 8 °C to 10 °C; PC = 50%) (4 stallions × 8 cycles) (P = 0.002). Sperm stored at −0.5 °C to 3 °C had lower acrosome integrity/responsiveness, similar membrane integrity (HOS test) and motilities, and higher VCL and ALH, than semen stored between 4 and 15 °C. These results demonstrate a wide tolerance of equine sperm to variable positive temperatures and air exposure for 22 h storage and more. However, temperatures close to 0 °C are detrimental for fertility.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

Long-term storage of Pennisetum glaucum (L.) R. Br. pollen

Summary Pearl millet [Pennisetum glaucum (L.) R. Br.] pollen has been successfully stored for 2,615 and 2,911 days at -18° and -73 °C, respectively, and continues to be viable. Viability of pollen stored at -73 °C appears to be little affected either by pollen storage moisture contents below 7.2% or by storage in glass vial or zip-lock plastic bag containers. Pollen moisture content appears to be more critical for maintaining viability at -18°C than at -73°C. Glass vials appear to be more desirable for longer term (>3 years) storage at -18°C.     

Viability and Storage of Pollen of the Oil Palm, Elaeis guineensis Jacq

The effects of drying pollen of the oil palm, Elaeis guineensis,and storage under various conditions, on its viability havebeen examined. Viability was based on the ability of pollento germinate on a sucrose medium with and without added boricacid, and on its capacity to set fruit when applied to receptivefemale flowers. Oven-drying at 37 °C for 2–8 h followedby storage in a deep freezer proved to be the best method ofstoring the pollen. Pollen treated in this way and stored for12 months was capable of producing fruits which were equal inweight and appearance to those produced by fresh pollen. Elaeis guineensis, oil palm, pollen, storage, viability     

Repressing the expression of self-incompatibility in crucifers by short-term high temperature treatment

Summary The effect of short-term high temperature on the expression of self-incompatibility was studied in detached flowers of Brassica oleracea, B. campestris and Raphanus sativus. The expression of self-incompatibility was repressed by treatment of pistils at 40 °C for 15 minutes. Treatment at 50 °C repressed self-incompatibility but it also disturbed pollen tube elongation into stylar tissue. S-glycoproteins did not show any quantitative changes during the intact pistil treatment under 50 °C. Callose was occasionally found in the treated papilla where the self pollen tube penetrated. The repressing effect of the 40 °C treatment was found to be reversible, and this reversibility depended upon the environmental temperature of plant. Plants grown at 15/5 °C (day/night temperature) completely recovered self-incompatibility 2 h after treatment, while those grown at 20/10°, 25/15 °C did not. The reversibility of the expression of self-incompatibility correlated with the distortion of plasma membrane in the papilla. It is considered that high temperature affects the pollen tube penetration system in pistils rather than the recognition system between pistils and pollen. The treatment of dehiscing anthers at 40 °C killed the pollen.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Physical factors influencing the production of strawberry aroma byPseudomonas fragi grown in skim milk

Summary The influence of temperature, agitation speed, pH and biomass on the synthesis of 19 metabolites contributing to a strawberry aroma was followed over a 72 h fermentation of skim milk byPseudomonas fragi. Amongst the major odor-active metabolites were ethyl butyrate, ethyl hexanoate, ethyl 2-hexenoate, ethyl crotonate, ethyl isovalerate and ethyl 2-methyl hexanoate. Up to 17 ppm of some of these metabolites were detected at 60 h of fermentation, approximately 36 h after the beginning of the stationary growth phase. The production of most odor-active metabolites was higher at 11°C and 150 RPM than at 15, 20 or 25°C and 200 or 250 RPM. The development of off-aromas was observed at high temperatures and at high agitation speeds.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

High activity xylanase from thermotolerantStreptomyces T7: Cultural conditions and enzyme properties

Summary A thermotolerantStreptomyces T₇ produced 70–72 U/ml of extracellular xylanase activity when grown at 50°C in submerged culture, in à medium containing 5% wheat bran as a carbon source. Among the various sugars tested, maltose showed the highest activity of 8 U/ml. Pure xylan was less effective as an inducer as compared to wheat bran. Ammonium sulphate at a concentration of 0.7% was found to be optimum for maximum yield of the enzyme. The optimum period and pH for maximum production were 72th and 7.0, respectively. The culture filtrate was devoid of amylase, cellulase and B-xylosidase activity. The xylanase was exceptionally stable and did not show any loss in activity after storage at 50°C at pH 5.0 for 6 days.     

Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field.     

Capsaicin hydrolysis by Candida antarctica lipase

Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase.     

Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study

Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 degrees C or 22 degrees C, while those diluted with skimmed milk were cooled at 15 degrees C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO2, 7% O2 and 88% N2) at 39 degrees C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 degrees C.     

Survival of Neozygites cf. floridana (Zygomycetes: Entomophthorales) in mummified cassava green mites and the viability of its primary conidia

The survival of Neozygites cf. floridana (Weiser and Muma) as dry hyphal bodies in mummified cassava green mites, Mononychellus tanajoa (Bondar), at 5.0% RH in the dark was affected by storage temperature. Survival of the fungus in mummies kept at 24±1.0°C could be demonstrated for 6–7 months. When stored at 4°C, the fungus sporulated from 90% of the mummies liberating an average of 186.9 primary conidia per mummy even after a storage period of 16 months, when the experiment was terminated. The temperature, humidity and light condition significantly affected the viability of primary conidia. The percent viability across all factors dropped from 98.4% after 0 h (beginning of the experiment) to 23.4% after a 1 h exposure to the conditions tested. Lower temperatures maintained higher viabilities with 86.3% of the conidia surviving after 18 h at 18°C, whereas almost all conidia died after 12 h at 33°C. Conidia survived less than 1 h when exposed to SDs (saturation deficit) of 2.0 mm Hg or higher at any tested temperature.     

Studies of the survival ofFusarium oxysporum conidia produced by submerged culture

Summary To study the survival of conidia ofFusarium oxysporum produced by submerged culture on malt extract, a harvesting process and different packaging and storage conditions have been tested. Conidia dried with talc and stored at +4°C preserve their viability after about 4.5 months.     

Plantlet production through high frequency somatic embryogenesis in long term cultures ofEucalyptus citriodora

A highly embryogenic culture ofEucalyptus citriodora was obtained by repetitive embryogenesis from somatic embryos cultured in the dark on a medium containing 500 mg/l each of glutamine and casein hydrolysate, 30 g/l of sucrose and 5 mg/l of 1-napthaleneacetic acid. Cultures retained morphogenetic ability for upto 36 months when maintained at 27°C by subculture at intervals of 4–5 weeks. The subculture period could be extended beyond 9 months if cultures were incubated at 10°C. On a hormone free medium incubated in light 50% of the embryos germinated to plantlets of which 70% survived when transferred to a sand and soil mixture.Abbreviations NAA 1-naphthal eneacetic acid NCL Communication No: 4480     

Effect of storage temperature during transport of ovaries on in vitro embryo production in Iberian red deer (Cervus elaphus hispanicus)

The aim of this work was to study the effect of storage temperature during the transport of ovaries on cleavage and blastocyst rates in Iberian red deer, because wild populations of this subspecies are usually far from laboratories. A total of 472 ovaries from 236 Iberian hinds were recovered and maintained in saline solution at 5-8 °C or 20-25 °C for 12 h. After storage, aspirated oocytes were matured with FSH/LH or EGF and the developed embryos were cultured with oviduct epithelial cells monolayer (OCM). A higher (P = 0.009) cleavage rate was obtained when the ovaries were stored at 5-8 °C. However, there were no differences between both storage temperatures in relation to the percentage of blastocysts obtained. Considering the management and production systems of Iberian red deer, this study provides important information about the ovary storage temperature during transport with the purpose of assuring an optimal in vitro embryo production.     

Growth conditions of a pectinolyticAspergillus fumigatus for degumming of natural fibres

Summary Optimum growth conditions forA. fumigatus strain 4 when citric pectin was the sole carbon source were at a temperature of 45°C, pH 4.0 and an incubation time from 36 to 42h. Under these conditions no cellulase activity was found. When orange pulp was the sole carbon source, optimum polygalacturonase activities were found when the fungus was cultured for 36 h at 45°C and a pH 3.0 to 4.5.