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BACKGROUND Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. OBJECTIVE The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two-dimensional difference gel electrophoresis (2D-DIGE). MATERIALS AND METHODS Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D-DIGE. RESULTS After thawing, the proportions of viable and acrosome-intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D-DIGE were different between stallions. DISCUSSION These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity. CONCLUSION The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine-rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance.

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time‐PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p, The recovery of the epididymal spermatozoa from dead animals, the cryopreservation and subsequent IVF are useful tools to rescue genetic material that otherwise would be lost, either from highly productive animals or from endangered species. This study was designed for development of suitable cryopreservation protocols for bovine epididymal sperm. We collected testes tissues from a local slaughterhouse and stored them overnight in the refrigerator. Semen was recovered from the epididymis and the effects of cryopreservation on the patterns of bovine epididymal sperm motility and gene expression were evaluated and finally, the fertilizing potential was tested by IVF.

Contents In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris–fructose citrate extender, and stored at 4°C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48 hr of storage, after storage for 96 hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p

The aim of the present study was to characterize the epididymal sperm of Spix's yellow-toothed cavy (Galea spixii) through two different recovery methods. Nine sexually mature males were euthanized and the complexes, testes–epididymis, were dissected. For each animal, one epididymis was processed by flotation method and the other was processed by retrograde flushing method, both using a TES-based buffered media. After recovery, we evaluated the sperm for motility, vigour, viability, functional membrane integrity and morphology. Morphometric data from the different sperm regions were evaluated by using an appropriate software. After recovery, both methods provide similar values for all the sperm parameters, aiming the recovery of more than 300 × 106 sperm, presenting >50% motile sperm, with normal morphology and functional membrane. The total sperm length in this sperm was 48.87 ± 0.1, and the sperm head presented 9.4%, on average. A notable characteristic was the prominent acrosome found in the G. spixii sperm. In conclusion, we demonstrate that either flotation or retrograde flushing methods are suitable for the recovery of sperm from cauda epididymis of Spix's yellow-toothed cavies.

Contents To support conservation strategies in wild species, simple but highly reproducible procedures of sperm cryopreservation are required for an application under field conditions. We used epididymal sperm of the domestic cat to optimize a sperm freezing procedure for felid species, particularly questioning the demand for sperm cooling to 4°C. We equilibrated sperm during slow cooling to only 15 or 10°C in a Tes–Tris–fructose extender with final concentrations of 4.7% (v/v) glycerol and 10% (v/v) of the water-soluble fraction of hen's egg yolk (low-density lipoproteins). Subsequently, sperm were frozen over liquid nitrogen. Total and progressive motility (mean ± SD) after thawing was 60.7 ± 8.6% and 53.9 ± 9.6% in samples cooled to 15°C or 61.6 ± 9.5% and 55.3 ± 9.9% in samples cooled to 10°C. Therefore, a one-step addition of glycerol to sperm at room temperature together with the freezing extender, the use of cryovials (loaded with diluted sperm aliquots of 300 μl), an equilibration period of 40 min comprising slow cooling to 15°C at a rate of approximately −0.14 K/min before rapid freezing over liquid nitrogen, yielded satisfying results. Cooling, freezing and thawing rates were exactly characterized as a prerequisite for further optimization and to provide a repeatable protocol to other practitioners.

Department of Morphology, Institute of Biosciences, UNESP - Univ. Estadual Paulista, Botucatu, Sao Paulo, Brazil.

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10(6) /mL) was lower (P 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone-stimulated Ban and Landrace. The percentages of two-cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non-stimulated Ban (4.0 ± 3.8%) was lower (P

Summary Sperm production rate was not adversely affected in rats which received over a period of eleven days three or six intraperitoneal injections, each of 12 · 50 μg of aflatoxin B1 is 1 % albumin. The toxin also failed to exert any change on the amount and types of abnormality among the epididymal sperm. Male fertility as evidenced by conception rate, litter size and embryo survival was not affected. The results suggest that the ingestion of Aspergillus-infected food resulting in the production of 50 μg of aflatoxin B1 per kilogram of body weight for a short time will not influence spermatogenesis, the sperm stored in the epididymides or fertility in the male. Zusammenfassung Einflus von kleinen Dosen von Aflatoxin B1 auf die Grose der Spermaproduktion, die epididymalen Spermienabnormitaten und die Fruchtbarkeit der Ratten Bei Ratten, welche wahrend 11 Tage 3 oder 6 Injektionen von je 12,5 μg Aflatoxin B1 in 1 % Albuminlosung erhielten, wurde die Spermienproduktion nicht nachteilig beeinflust. Auch die Anzahl abnormer Nebenhodenspermien wurde durch das Toxin nicht verandert. Die Fruchtbarkeit der Rattenmannchen, gepruft an Hand der Konzeptionsrate, Wurfgrose und Uberleben der Fruchte, wurde ebenfalls nicht beeintrachtigt. Die Ergebnisse sprechen dafur, das die Aufnahme Aspergillusinfizierten Futters (50 μg Aflatoxin B1 / kg Korpergewicht) wahrend kurzer Zeit weder die Spermatogenese noch die Spermienspeicherung im Nebenhoden und Fertilitat der Mannchen reduziert. Resume Influence de petites doses d'aflatoxine B1 sur le volume de production du sperme, sur les anomalies des spermatozoides de l'epididyme et sur la fertilite des rats La production des spermatozoides n'a pas ete influencee defavorablement chez des rats qui avaient recu durant 11 jours 3 ou 6 injections de 12,5 μg d'aflatoxine B1 chacune dans une solution d'albumine a 1 %. Le nombre de spermatozoides anormaux de l'epididyme n'a pas ete modifie par la toxine. La fertilite des rates mâles testee sur la base du taux de conception, de la grosseur des portees et de la survie des jeunes n'a egalement pas ete influencee. Les resultats indiquent que l'absorption d'un aliment contamine par Aspergillus (50 μg aflatoxine B1 / kg de poids) durant un court laps de temps ne reduit ni la spermatogenese, ni la production des spermatozoides dans l'epididyme, ni la fertilite des mâles. Resumen Los efectos de microdosis de aflatoxina B1 sobre las tasas de produccion de esperma, las anormalidades de los espermatozoides del epididimo y la fertilidad de los ratasmachos La tasa de produccion espermatica no fue afectada de forma adversa en ratas-machos que habian recibido durante un periodo de once dias tres o seis inyecciones intraperitoneales, cada una de 12,5 μg de aflatoxina B1 en solucion de albumina al 1 %. A igual, no fue modificada por la toxina la cantidad de espermios epididimales anormales. Tampoco resulto afectada la fertilidad de los ratas-machos, evidenciada a la vista de la tasa de concepcion, tamano de la camada y supervivencia de las crias. Los resultados abogan a favor de que la ingestion de pienso infectado por Aspergillus (50 μg de aflatoxina B1/kg peso en vivo) durante un espacio breve de tiempo no reduce ni la espermatogenia ni el almacenamiento de espermatozoides en el epididimo ni la fertilidad de los machos.

The effects of exposure to constant light (CL) and blinding on male reproductive behavioral physiology of South Indian gerbils (Tatera indica cuvieri) were assessed. Exposure to CL diminished reproductive efficiency of males with a reduction in the proportion of ejaculating males. This is further evidenced by increase in the number and duration of intromissions and intromission latency. Concomitantly, reproductive organ weight and epididymal sperm count were also reduced. However, in weanlings CL did not induce changes either in their maturational process or their reproductive organs’ weight. Blinding of adult reproductively inactive males resulted in a considerable proportion of them exhibiting sexual activity, which is reflected in the seminal vesicle weight and epididymal sperm count. Blinded weanlings showed earlier testicular descent and higher seminal vesicle weight. These studies reveal that blinding (constant darkness) has a stimulatory effect whereas CL is inhibitory in the reproduction of the tropical rodent Tatera indica cuvieri. J. Exp. Zool. 289:59–65, 2001. © 2001 Wiley-Liss, Inc.

Inhalt Folliculare unreife Oozyten der Hauskatze wurden hinsichtlich ihrer Befruchtungs-und Entwicklungskompetenz unter dem Einfluβ von felinen Oviduktepithelzellen (FOEC) untersucht. Nonovulatorische Oozyten wurden durch Anschneiden der Oberflache ektomierter Ovarien und Ausspulen der Follikel gewonnen. Eine Klassifizierung der Oozyten erfolgte nach der optischen Dichte des Vitellus. Mit epididymalen Spermien, deren wichtigste Parameter ausgewiesen werden, wurde inseminiert. Oozyten mit optisch dichtem Vitellus zeigten eine viermal hohere Befruchtungs- und Teilungsrate als Oozyten mit transparentem Vitellus. Eine Kokultivierung von FOEC wahrend des Inseminationsvorganges und der Embryokultivierung fuhrte sowohl zu einer signifikanten Steigerung (p < 0,05) der Befruchtungsrate von 34,9% auf 49,5% als auch der Furchungsrate von 30,3% auf 48,6%. Contents: Developmental competence of domesticcatfollicular oocytes after fertilization in vitro with epididymal sperm and co-culture of feline oviductal epithelial cells. Follicular immature oocytes from ectomized ovaries of the domestic cat were tested on fertilization and cleavage competence during influence of feline oviductal epithelial cells (FOEC). Oocytes were liberated by cutting the surface of the ovary. The oocytes were selected into two groups: class-I-cumulus-oocyte-complexes (COC's) with a dark and class-II-COCs with a transparent vitellus. The COC's were cultured for 48 h in an complex medium (TCM199) with 0,3% bovine serum albumin. Epididymal sperm were used for insemination. Class-I-COC's achieve four times higher rate infertilization and cleavage than class-II -COC's. During the co-culture of FOEC both the rate of fertilization and of cleavage are facilitated significantly (p < 0,05) from 34,9% to 49,5% and from 30,3% to 48,6%, respectively.

An aqueous extract of Cynomorium coccineum was administered by stomach tube to ten mature male Wistar rats, at a dose of 47 mg/100 kg body weight/day for 14 consecutive days. Ten rats were kept as controls and received normal saline by oral route at the same dosing interval. Sperm was collected from the epididymes after decapitation. The results revealed that the water extract of the Cynomorium coccineum induced significant increase in the sperm count, improved the percentage of live sperm and their motility and decreased the number of abnormal sperm. Testicular histology showed increased spermatogenesis and seminiferous tubules full of sperm in the treated group compared with the controls.

Making Epididymal Sperm Counts (Yefan Wang, TherImmune Research Corporation, Gaithersberg, Maryland). Epididymal sperm counts are a widely used, simple and sensitive method of assessing the effects of male reproductive toxicants on the epididymal and/or testicular site of action. After careful dissection of the tissues and further processing, the sperm suspensions are counted using a hemacytometer and analyzed for effect.

Motility characteristics of epididymal sperm from tw32/+ and T/+ males of several strains were compared to those of sperm from the congenic, wild, type mice. Epididymal sperm from C57BL/6-tw32/+ (B6-tw32/+) males showed a significant decline in vigorous motility and a significant increase in adhesivity to glass during 7.5 hours in vitro, while sperm from B6-+/+ males showed no changes in these characteristics. The apparent mean velocities of epididymal sperm were: B6tw32/+, 32 micro/sec; C3H-tw32/+, 60; B6XC3H hybrids (F2*-tw32/+), 44; and C3HXB6 hybrids (F2-tw32/+), 58. Each of these values was significantly lower than that of sperm from their congenic wild-type strains: B6- +/+, 115; C3H- +/+, 177; F1*-+/+, 168 and F1-+/+, 195 micro/sec. B6-T2J/+ sperm had velocities intermediate to the congenic +/+ and tw32/+ genotypes, whereas C3HT/+ sperm velocity was not significantly different from C3H- +/+. These differences in measured sperm velocity may not reflect real differences in swimming speed, but rather may indicate a difference in the shape of swimming trajectories. Erratic and convoluted swimming trajectories were observed for the majority of sperm from tw32/+ males of all genotypes. Differences in motility patterns of sperm from tw32/+ males could play a role in the abnormally high transmission of tw32 through the male.

Estimation of sperm concentration with ethidium bromide was found to be simple and rapid. The estimated value of DNA per human spermatozoon, 2.7 pg, agreed with published values. The method is also applicable to rat epididymal spermatozoa, although the apparent concentration of DNA is only 1.5 pg/spermatozoon. In spectrophotometry, falsely high values were noted. These were, however, reproducible and did not interfere with sperm estimation. Although fluorometry is not more sensitive than spectrephotometry, it is simpler, avoiding corrosive reagents, and more rapid. A standardized method is outlined and calibration equations, based on correlation analysis, are presented.

Changes in the concentration, motility and morphology, of epididymal sperm have been evaluated in males of between 40 and 100 days of age in BALB, CBA, C3H and C57BL strains of mice. Particular attention was paid to sperm morphology, and a multiple entry classification system was used for its quantitation. At 40 days in each of the 4 strains studied sperm were few in numbers with poor motility and highly atypical morphology, particularly in the head region. From 80 days of age values for the 3 parameters were within the normal range. At equivalent ages during puberty the 4 strains differed in the concentration and motility of epididymal sperm, indicating difference in the time of onset of puberty between the strains. However, once adulthood was reached the strains differed only in the percentage of normal forms and in the frequency distribution of sperm with the various morphological abnormalities.

Conditions were established for routine cinemicrographic examination of sperm incorporation by living zona-free mouse eggs employing oil immersion objectives and Nomarski optics. Initial sperm attachment to the egg plasma membrane, which was reversible and appeared to require flagellar activity, involved localized areas of the head corresponding approximately to the position of the equatorial segment. Penetrating sperm lay flat on the egg and, during incorporation, appeared to sink into the egg cytoplasm, accompanied by short bursts of flagellar activity and subsequent rotation of the flagellum around its insertion point. Ensuing sperm head decondensation involved dissociation of individual particulate structures and a dramatic localized clearing in the egg cytoplasm. The normalcy of the penetration process and the potential applicability of this approach was attested to by the observations that polar body extrusion, male and female pronuclear formation, and migration through the egg cytoplasm in preparation for syngamy occurred in several sequences followed for extended time periods.

— The methods and apparatus for accomplishing artificial insemination in chinchilla are described along with pertinent observations and discussion. This general subject is treated under the following topics: causes of sterility and means of alleviating some forms; anaesthetics and induction of anaesthesia; methods of ejaculating the male; sperm buffer media; evaluation of semen; technique of inseminating the female; surgical preparation of the vesiculectomized male for epididymal sperm.

Fertilization after tubal insemination with epididymal sperm in the rabbit Sperm from the distal corpus and proximal cauda epididymides was introduced into the ligated oviduct of females treated with HCG to induce ovulation. Of the ova recovered 50% (distal caput) and 94% (proximal cauda) respectively, were fertilized.