Prolonged survival of fowl spermatozoa stored in a diffusion chamber in vitro

1. A diffusion chamber technique was used in an attempt to prolong the life-span of fowl spermatozoa at 41 degrees C in vitro. 2. The most suitable conditions in the diffusion chamber system for this purpose were found to be pH 7.2, with the addition of foetal calf serum (400 mg/ml), a sperm concentration of 10 X 10(9)/ml and a gas mixture of 5% CO2 + 5% O2 + 90% N2. 3. The spermatozoa stored in the diffusion chamber under these conditions were highly fertile for at least 2 d, whereas those stored in a test tube had a low fertilizing ability.     

Quantitation of the fertilising ability of fresh compared with frozen and thawed fowl spermatozoa

The fertilising abilities of freshly-diluted and frozen and thawed fowl semen were compared by inseminating a wide range (0.2 to 1600 X 10(6] of doses of spermatozoa. These data demonstrated that there was a non-linear relationship between numbers of spermatozoa and the probability of fertilisation. It was concluded that a typical fertility trial, which assesses the percentage of fertile eggs laid by groups of hens inseminated with a fixed dose of semen, was inadequate for comparing the quality of semen samples. An alternative fertility trial is described, which involves assessing the numbers of spermatozoa required to fertilize a particular percentage of eggs laid. This modified method showed that the quality of frozen and thawed semen, in terms of its fertilising ability, was reduced to 1.6% of that of fresh semen.     

Effect on fertility of low numbers of fowl spermatozoa inseminated in aqueous diluent or semen components of the fowl and turkey

A study was undertaken to compare the effect on fertility in the fowl of aqueous medium, natural homologous seminal plasma, heterologous turkey seminal plasma and whole turkey semen when whole fowl semen was excessively diluted with these media and inseminated fresh. High dilution with fowl seminal plasma resulted in the best fertility. Dilution with the turkey semen components produced fertility no different from that with aqueous diluent when the dose of spermatozoa was 5 X 5 or 10 X 10(6). The results of this study confirm that 5 X 5 to 10 X 10(6) good quality spermatozoa are sufficient to produce acceptable fertility in weekly inseminations of fresh semen. This enables good quality semen to be highly diluted. However, at high dilution rates there is a need to reconsider the composition of semen diluents, with respect to simulating as yet unknown properties provided by factor(s) in homologous ductus deferens seminal plasma.     

'Serum like' albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4 degrees C.

1. Using immunoelectrophoretic and immunodiffusion methods, serum-like albumin was detected in fowl seminal plasma. Immunodiffusion showed seminal plasma albumin concentration to be 4 mg/ml, corresponding to half of the total proteins (8 mg/ml). 2. Replacing seminal plasma with a diluent containing either 1, 4, or 16 mg/ml albumin increased motility of spermatozoa stored for 24 h at 4 degrees C, 16 mg/ml being the more effective dose. 1 and 4 mg/ml had no effect on the fertilising ability of fowl spermatozoa stored for 24 h at 4 degrees C in both young (28-35 weeks old) and old birds (50-55 weeks old). 16 mg/ml albumin had no effect on fertilising rates in young but depressed it in old birds. 3. These results indicate that seminal plasma albumin may be one of the mobility stimulating factors of seminal plasma. However it does not protect fertilising ability better than the diluent alone.     

Effects of ovalbumin on the motility and fertilising ability of fowl spermatozoa stored for 24 H at 4 degrees C

1. The effects of 0.1, 0.5, 1 and 3 g ovalbumin/100 ml diluent on spermatozoa stored for 24 h at 4 degrees C in TES diluent or BPSE diluent with or without seminal plasma were studied. 2. There was no effect of ovalbumin on the motility of spermatozoa stored in whole semen, except at an incorporation rate of 3 g/100 ml, when motility was reduced for spermatozoa stored in TES diluent. When sperm were stored in the absence of seminal plasma, ovalbumin stimulated motility but this effect was transitory. 3. The effects of ovalbumin on fertilisation rates were diluent- and concentration-dependent. Ovalbumin concentrations of 0.1 and 0.5 g/100 ml increased the fertilising ability of whole semen stored in TES diluent but 1 g/100 ml ovalbumin decreased it. However, 0.5 g ovalbumin/100 ml had no effect on the fertilising ability of spermatozoa stored in BPSE diluent, irrespective of the presence or absence of seminal plasma.     

Physiological changes in fowl and turkey spermatozoa during in vitro storage

1. Although early work on semen storage has been rather empirical in approach, only basic research can provide a framework of biological mechanisms from which improvements in the techniques of cryopreservation and liquid semen storage can progress logically. 2. A major drawback in this work has been the lack of adequate tests for quantitating and differentiating aspects of 'fertility'. 3. Basic research has now provided techniques for assessing: sperm fertilising ability in terms of numbers of fertile eggs; the efficiency of hens' oviducts at accepting and retaining spermatozoa, and sperm 'quality' as motility, metabolism and plasma membrane patency. 4. These techniques may be used for a more critical assessment of the effects of both cryopreservation and liquid semen storage on sperm function, although the integrity of sperm surface proteins may be a more sensitive variable which has yet to be measured. 5. Further improvements in sperm cryopreservation technology are best approached through an understanding of the fundamental cellular and molecular changes which take place during freezing; thus far little is known of such changes in avian spermatozoa. 6. The ideal milieu for maintaining spermatozoa in liquid semen storage should mimic the environment of the oviducal sperm storage tubules; elucidation of the factors involved in progressing steadily.     

Fertility results after insemination of a moderate count of cock sperm, diluted in Blumberger cock sperm diluent and homologous seminal plasma

Incomplete studies into artificial insemination of White Leghorn hens appear to suggest that the generally common number of cock spermatozoa per insemination can be considerably reduced without adverse consequences for reproduction results. Fertilisation rates between 89.4 and 94.9% were recorded from inseminations of as little as 20 x 10(6) spermatozoa, based on tenfold dilution of ejaculate collections in Blumberg Cock Sperma Diluent (BCSD) or homologous seminal plasma or differentiated combinations of both. Addition of increasing amounts of seminal plasma, however, caused decline in hatching rates as a result of rising embryonic mortality. Best suitability was recorded from a diluent combination of 75% of BCSD with 25% of seminal plasma, with the hatching rate being 85.0%.     

Prolonged survival of fowl spermatozoa in the oviducal tissues in organ culture

1. When fowl spermatozoa were incubated at 41 °C with tissues from the infundibulum, magnum, isthmus, shell gland, uterovaginal junction and vagina, their motility, assessed at room temperature (20 to 25 °C), was maintained for 4 to 12 d.

2. The survival period was much longer for spermatozoa incubated with the tissues from the infundibulum and uterovaginal junction (with sperm‐host glands, 11 to 12 d) than for those incubated with tissues from the other regions (lacking host glands, 4 to 7 d).

3. In the tissues of the infundibulum and uterovaginal junction, spermatozoa entered the sperm‐host glands and were more closely associated with the epithelial cells than they were in tissues from other regions.     

Evaluation of pentoxifylline and Basal Medium Eagle supplemented to diluent on cryopreserved goat spermatozoa

This study investigated the effect of pentoxifylline (PTX) and Basal Medium Eagle (BME) on frozen–thawed goat spermatozoa. Immediately after initial examination of ejaculated semen, samples were pooled and reexamined for quality. Then, samples were divided into eight equal aliquots and diluted with a basic tris-extender containing PTX (3, 6, 9 mM) and BME (5 mM) to reach a final concentration of 25 × 10⁹ and frozen. After 24 hr, the samples were individually thawed at 37°C for 30 s and evaluated for different characteristics. Obtained post-thaw results from Computer-Assisted Sperm Analysis indicate using of 3 and 6 mM PTX led significantly to an improvement in total motility, progressive motility and velocity characteristics of spermatozoa, except the beat/cross frequency (BCF) which indicated statistically no differences (p > .05) among control and treatments. Diluents prepared with BME (5 mM) and PTX alone (3 and 6 mM) improved significantly the membrane integrity–functionality, acrosome integrity and also hyaluronidase activity. Regarding recovery rate, the results showed significantly (p < .05) higher values for diluents containing 3 and 6 mM PTX compared to other groups. Malondialdehyde concentration exhibited also a significant difference (p < .05) in diluents supplemented with 5 mM BME, 3, 6 and 9 mM PTX, and mixture of 3 mM PTX and 5 mM BME which illustrate a similarity for active mitochondria, apoptotic-like and dead spermatozoa. Finally, the ratio of sperm chromatin dispersion stained spermatozoa presented significant differences (p < .05) among treatments in which the diluents added PTX alone demonstrated significantly lower values than control and extenders containing the mixtures of BME and PTX. In conclusion, the observation in this study indicates using of 3 and 6 mM PTX and BME alone may improve significantly (p < .05) the quality of cryopreserved goat spermatozoa.     

Fertility,hatghability and malformations in guinea fowl embryos as affected by dietary manganese

1. The effects of feeding diets containing 54 mg, 60 mg, 65 mg or 70 mg manganese/kg to Guinea fowls on fertility, hatchability and the occurrence of malformed embryos were studied.

2. Dietary manganese affected fertility slightly, but significantly affected hatchability and the occurrence of malformed embryos.

3. Hatchability was most significantly depressed at the lowest dietary concentration of manganese, which also caused a highly significant increase in the proportion of malformed embryos.

4. Although increasing manganese to more than 54 mg/kg improved hatchability and reduced embryonic malformations, the increase did not completely eliminate the latter condition.     

Microbiological examination of fresh and stored procusts of cold cuisine]

In four out of five investigated kinds of products it was proved that most fresh samples met the microbiological requirements of Czechoslovak State Standard CSN 58 3601 and it is even feasible to consider a stricter approach to the evaluation of the total number of microbes [CPM] to 10(5) per g and in the number of coliform microbes to 10(3) per g. On contrary the amount of yeast in most fresh samples [62%-100%] exceeded the permitted limit and reached the value of about 10(4) per g. The comparison of the results of the investigation of fresh and stored samples indicates that from the eight groups or kinds of microorganisms under study the highest rate of reproduction was in yeast. In the other microorganisms the increase was not so great nor did it occur. No pathogenous microorganisms of salmonella were determined by the orientation method, and staphylococci was proved by direct cultivation in just one kind of product.     

Association of (3H) DNA with fowl spermatozoa and their in vitro fertilisation of hamster ova.

1. Fowl spermatozoa incubated with DNA were used for the fertilisation of zona-free hamster oocytes. 2. Two categories of spermatozoa were detected: those which were not labelled and those which showed a very high degree of labelling (19 +/- 2% of the whole population). 3. In the hamster oocytes exposed to treated spermatozoa the labelled sperm heads were identified.     

Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing

In order to investigate the factors causing crooked-necked spermatozoa (CNS) or those with abnormal acrosomes during freezing and thawing, fowl spermatozoa in NaCl or glucose solutions containing 92 ml glycerol/l were examined using a scanning electron microscope before and after freezing and thawing. The incidence of CNS in NaCl solution significantly increased after freezing and thawing, but not in glucose solution. The acrosomal damage caused by freezing and thawing was considerable in both solutions, and the incidence of damage in glucose solution was significantly higher than that in NaCl solution. In neither solution was there a significant difference between the incidence of acrosomal damage in CNS and in non-CNS. The ratios of incidences of abnormal acrosome after, versus before, freezing were higher in non-CNS than in CNS. It appears from these results that the factors during freezing and thawing which cause CNS may differ from those causing acrosomal damage.     

Piezo-assisted in vitro fertilization of mouse oocytes with spermatozoa retrieved from epididymides stored at 4 degree C

This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa.     

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa

Metformin is clinically used to treat diabetes. Given its role‐impacting metabolism, metformin has been also added to semen cryopreservation media showing specie‐dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin‐including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ‐population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.