Effect of low and high egg yolk concentrations in the semen extender for goat semen cryopreservation

This research aimed to evaluate two concentrations of egg yolk inclusion rates (20 and 2.5%) in the semen extender of goat semen cryopreserved during two seasons of the year. The study was conducted during a light-induced breeding season (Experiment 1), and during the natural breeding season (Experiment 2), in the southern hemisphere. Four ejaculates from each buck (n = 2) were collected in each experiment. After collection, semen was divided, with each sample being diluted in the semen extender – according to the treatments (T1 – 20% egg yolk or T2 – 2.5% egg yolk, using a glucose–EDTA extender). For T1 treatment in Experiment 2, the semen was also washed before the semen cryopreservation process. The semen samples were frozen, and after thawing evaluated for seminal characteristics i.e. sperm motility, vigor, morphology and membrane integrity. The fertilising capacity of the frozen-thawed semen was evaluated following a single artificial insemination 12 h after the onset of estrus in 50 (Experiment 1) and 60 does (Experiment 2). In Experiments 1 and 2, the mean values for sperm motility and membrane integrity of the frozen-thawed semen did not differ between the T1 and T2 treatments. However, the mean sperm vigor and morphological normal sperm were greater (P < 0.05) in T2 than T1 treatment. The fertility rates recorded did not differ between T1 and T2 treatments in Experiment 1, however, it was greater (P < 0.05) in the T2 than in the T1 treatment, in Experiment 2. According to obtained results, it can be recommended to use a glucose–EDTA extender with a low egg yolk concentration (2.5%) inclusion, for superior fertility results in goats.     

Development of a chemically defined ram semen diluent (RSD-1)

A chemically defined ram semen diluent (RSD-1) has been developed. RSD-1 maintained spermatozoal motility of diluted semen containing approximately 800 million spermatozoa ml⁻¹ during cooling to 15°C and its storage for 1 h. Motility was further maintained when the cooled semen was diluted to 100 million spermatozoa ml⁻¹ and incubated at 38°C for about 24 h. In contrast, a conventional milk-based diluent supported motility for less than 6 h at 38°C. Spermatozoal motility was influenced by the buffering capacity, osmolarity and the presence or absence of macromolecules and calcium in the chemically defined diluent. Among the organic buffers tested, MOPS (3-(N-morpholino)propanesulphonic acid) had a marked influence on the maintenance of spermatozoal motility. The presence of MOPS also overcame the detrimental effects of 2 mM calcium in Krebs Ringer improved (KR-I) buffer.     

Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.     

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.     

Effect of glycerol level in Tris-basek extender and equilibration period on quality of frozen goat semen

Twenty ejaculates, 4 from each of 5 native goats, were collected using an artificial vagina, and the effects of glycerol level (4, 6.4 and 9 %) and the equilibration period (1, 3 and 5 h) were studied by split-sample technique. The extender used was Tris egg yolk citric acid fructose glycerol extender. The semen was frozen in 0.5-ml French straws by exposure for 10 min to liquid nitrogen vapor, 5 cm above the liquid nitrogen level. After 14 h of storage in liquid nitrogen, the straws were thawed in water at 37 degrees C for 12 - 15 sec. The percentage of progressively motile sperm (PPM) and the percentage of damaged acrosomes (PDA) were studied after equilibration and after thawing. The mean PPM after thawing was found to be 64.0 +/- 0.90, 66.92 +/- 0.54 and 63.65 +/- 1.07 when semen was frozen with 4, 6.4 and 9 % glycerol and 61.48 +/- 0.81, 65.05 +/- 0.78 and 68.03 +/- 0.87 in 1-, 3- and 5-h equilibrated semen, respectively. The mean PDA after thawing was 7.12 +/- 0.88, 8.23 +/- 0.76 and 10.58 +/- 0.84 when semen was frozen with 4, 6.4 and 9 % glycerol and 7.0 +/- 0.74, 9.0 +/- 0.95 and 9.93 +/- 0.81 in 1-, 3- and 5-h equilibrated semen, respectively. Both PPM and PDA differed significantly (P<0.01) between glycerol levels, between equilibration periods and between stages (after equilibration and after thawing). The PPM also differed significantly due to equilibration period x stage interaction (P<0.01) and glycerol level x stage interaction (P<0.05). The PDA did not differ significantly due to interactions. When the differences between pairs of means were tested by least significant difference, it was found that after equilibration PPM was not significantly affected by either glycerol level or equilibration period, while after thawing, it was significantly higher (P<0.05) for 6.4 % glycerol and 5-h equilibrated semen than for 4 or 9 % glycerol and 1- or 3-h equilibrated semen, respectively. The PDA was lower with 4 % glycerol and 1-h equilibrated semen.     

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 μM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 μM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.     

Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25 degrees C min(-1) to 5 degrees C, 5 degrees C min (-1) to -20 degrees C, 20 degrees C min(-1) to -100 degrees C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0-18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me(2)SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.     

Induction of the acrosome reaction in ejaculated goat spermatozoa by preincubation in chemically defined medium

The aim of the present study was to examine the effects of the elimination of energy substrate from the medium and the effects of the preincubation vessel, temperature, and time on the induction of the acrosome reaction in ejaculated goat spermatozoa in chemically defined medium. Washed spermatozoa were resuspended in Brackett-Oliphant (BO) medium or substrate-free BO medium to give a high concentration and preincubated in open test tubes or sealed glass tubes at 37.0 or 39.5 degrees C for 1, 2, or 3 h. Sperm acrosome reaction was evaluated using a simplified triple-stain technique and a hamster test. It was found that the goat sperm acrosome reaction occurred more readily in the sealed glass tubes than in open test tubes, in substrate-free BO medium than in BO medium, and at 39.5 than at 37.0 degrees C. During preincubation with substrate-free BO medium in sealed glass tubes at 39.5 degrees C, the optimum time necessary to induce the acrosome reaction in goat spermatozoa was 2-3 h.     

Characteristics and seasonal variations in the semen of Alpine, Saanen and Damascus goat bucks born and raised in Greece

Twenty-three purebred Alpine (n=8), Saanen (n=7) and Damascus (n=8) goat bucks raised at the Institute of Reproduction and Artificial Insemination in Thessaloniki, Greece (40 degrees 37 minutes N, 22 degrees 58 minutes E and altitude 32 m above sea level), were used to study the effect of photoperiod on semen production. Samples were collected with an artificial vagina and examined immediately after collection. In spite of the variation in nearly all semen characteristics studied among the 3 breeds of bucks, there was significant seasonal variation in both semen quantity (volume, concentration and total number of spermatozoa per ejaculate) and quality (percentage of motile spermatozoa, percentage of abnormal spermatozoa and rate of progressive motility). The best semen was produced during the breeding season (late summer and autumn). However, the magnitude of these seasonal effects was not sufficient to prevent bucks from being used for breeding throughout the year. Nevertheless, individual differences in the semen quantity and quality among bucks within a breed make individual evaluation of semen necessary to select the most fertile males for breeding.     

Comparison of ticarcillin and piperacillin in Kenney's semen extender

Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial concentration. All three solutions were stored at 4 degrees C and aliquots were obtained at 24 and 48 h to determine sperm motility and microbial concentration. Mean percentages of motile and progressively motile sperm did not differ significantly among control and antibiotic-containing solutions after storage. Control-extended semen samples from ejaculates of stallions (n=11) were contaminated with aerobic gram-positive and gram-negative bacteria. In solutions that contained either antibiotic, growth of these microbes was inhibited after 1, 24, and 48 h at 4 degrees C. Semen samples from stallions (n=5) were extended with Kenney's glucose skim milk extender containing no antibiotic, ticarcillin or piperacillin and then inoculated with approximately 5 x 10(2)CFU/mL Klebsiella pneumoniae or Pseudomonas aeruginosa; there was no significant difference between antibiotics in the inhibition of microbial growth. In conclusion, piperacillin was an appropriate alternative to ticarcillin in extenders for equine semen.     

Cultivation of Pasteurella haemolytica in a chemically defined medium

A chemically defined medium was developed for the aerobic cultivation of Pasteurella haemolytica. Studies on the growth of strain H44L were conducted in a medium consisting of 15 amino acids, inorganic salts, citrate, nicotinamide, pantothenate, thiamine or thiamine monophosphate, and carbon sources. The amino acids were provided as l isomers, because racemic mixtures of some amino acids inhibited growth. The carbon source consisted of a mixture of 1.0% d-galactose and 0.1% d-glucose. Culture populations of strain H44L reached 2 x 10(10) cells per milliliter after 16 hr of incubation at 37.5 C. Other strains of P. haemolytica, from a wide variety of sources, were tested for growth in the medium, and 23 of 24 strains grew well. Five strains of P. haemolytica var. ureae failed to grow in the medium.     

Development of a chemically defined diet for ants

A chemically defined diet is a useful tool for the study of nutritional physiology of organisms. We have developed such a diet for Camponotus carpenter ants to facilitate experiments on nutritional requirements of these ants. Worker colonies of Camponotus floridanus were fed with a chemically defined diet, containing all essential minerals, amino acids, vitamins, growth factors and sucrose in an agar matrix. After 13 weeks, neither the number of raised pupae, their dry weight, nor the mortality of workers in subcolonies fed with this diet differed significantly from control colonies fed with Bhatkar-Whitcomb-agar, in addition to cockroaches and diluted honey. Therefore, this diet is adequate for a normal brood production and a maximal growth rate of C. floridanus larvae, at least for a period of three months. This diet should be suitable for ants that are able to feed on agar-based food resources in general. Received 6 September 2006; revised 5 October 2006; accepted 18 October 2006. An erratum to this article is available at .     

Embryonic stem cell development in a chemically defined medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

Effects of cooling and freezing on pH of semen extender

The pH change of 10 different buffering systems with temperature ranging from room to 5 °C was examined; three were conventional buffers which included phosphate yolk, citrate yolk, and skim milk. Seven were Good's buffers with egg yolk which included TES, TRIS, BES, MOPS, PIPES, MES, and TEST. The pH of the three conventional buffers did not change with decreasing temperature, but Good's buffers showed an increase in pH with decreasing temperature from room to 5 °C. The pH change due to temperature was measured for TEST buffer solution with and without 20% egg yolk containing 2 or 6% of five different cryoprotective compounds. The pH at 5 °C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of egg yolk indicating that the change in pH is primarily due to the buffer. The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured continuously from 37 °C to below freezing (?18 °C). The pH increased with decreasing temperature to 8.0 ± 0.2 from 37 to ?14 °C at which point it dropped abruptly to pH 6.5 ± 0.2.