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International audience; Abstract Background Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal ( n = 19) or abnormal ( n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with (“H+” arm) or without (“H-” arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. Results Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p

The aim of the study was to evaluate the effect of repeated semen collection and the substitution of normal egg yolk with clarified egg yolk to commercially produced semen extender on qualitative parameters of frozen-thawed canine semen. Two semen collections were scheduled in a 24-hour interval and in each of six dogs, three 1st and three 2nd collections were performed. The frozen-thawed sperm samples were prepared either with clarified or normal egg yolk and motility and viability were evaluated. The effect of the sequence of semen collection was demonstrated by significant differences in motility and also in viability of sperms both in native and frozen-thawed ejaculate. The percentage of viable sperms was significantly higher in samples from the 2nd compared to the 1st collection. This trend was the same also in motility except in native ejaculate. The addition of clarified egg yolk was beneficial for higher survival of sperms immediately after thawing and also after 30 min of incubation, compared to samples with normal egg yolk. Sperm motility evaluated after thawing was higher in samples with clarified egg yolk, without an apparent connection with semen collection sequence. The decrease of values of the qualitative parameters of sperms observed in the period of 30 min of incubation was significantly slowed down when clarified egg yolk was used. This was especially obvious in samples from the 2nd collection.

Steroidal glycosides possess antioxidative endogenous properties on the peroxidation processes in biological products. Therefore, steroidal glycosides could represent one of the solutions for maintaining of morphological and physiological functions of spermatozoa. The researches aimed to study the influence of the steroidal glycosides as antioxidants, on cock sperm sanogenity in the cryoconservation process. The following physiological and morfological indices of sperm were assessed: the mobility, the pathological forms and acrosome integrity of spermatozoa. The obtained results established the optimal concentration of the -Lilia-Hand Treozida Lilia in the composition of synthetic media used for cock sperm cryoconservation. The antioxidative effect of the researched steroidal glycosides was confirmed by maintaining the morphological-functional status of the spermatozoa and diminution of the gamete pathology.

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol. © 2017

In the present study, we examined several cryoextenders previously used by several authors and various freezing protocols to determine the relative importance of each parameter on sperm freezing. The effects of controlled seeding and changes in cooling rate at different stages of freezing were also examined. Sperm samples from seven individual carp males were frozen in 0.5 ml straws by conventional freezing. Cooling rates were determined by monitoring the sample's internal temperature. We compared four freezing protocols, which involved placing sperm samples at various levels (1, 3, 6, and 9 cm) above the liquid nitrogen (LN) surface (corresponding to -190, -150, -110, and -70 °C, respectively) for 20 min followed by transferring the samples into LN. Freezing at 3 cm above the LN surface resulted in the highest motility (33 ? 8 %) and velocity (118 ? 9 ?m/s) of spermatozoa after thawing and diluting in swimming medium. We determined that -90 °C is an optimal temperature at which immersing the samples in LN does not affect sperm motility after thawing. The sperm motility of samples immersed in LN before or immediately after the crystallisation point (-16 °C) was 0 %. Motility of spermatozoa cryopreserved with or without a seeding procedure was not significantly different after thawing. Therefore, we hypothesise that supercooling the sample during the conventional freezing procedure is not the main damaging factor during carp spermatozoa cryopreservation.

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13 +/- 2 hours post fertilization: hpf), early D-larvae (24 +/- 2 hpf) and late D-larvae (43 +/- 2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13 +/- 2 hpf and at 43 +/- 2 hpf was significantly higher (P

The morphological and physiological characteristics of the testes and the sperm of the catfish #Heterobranchus longifilis$ (Val.) are presented. The effect of cryopreservation on the fertilizing capacity of the sperm was also evaluated. Testicular structure and spermatogenesis are described using histological techniques. The coexistence in the lobules of spermatozoa and all the spermatogenic stages indicates that this species is able to perform continuous reproduction. No seasonal trend was noticed in the evolution of the gonadosomatic index and in the quantity of the sperm produced over a year's period. However, maximum sperm production was observed in April and September. Different cryopreservation trials were conducted using a cryoprotective solution composed of 5% dimethylsulphoxide (DMSO), 5% glycerol, 10% hen's eggyolk and 80% Mounib's solution. Fresh and cryopreserved semen gave equivalent hatching rates (81.1%, 83.4% and 78.9% respectively for the fresh, the 1-hour cryopreserved and the 8-month cryopreserved sperm). Percentages of normal and deformed larvae were not affected by sperm cryopreservation. (Résumé d'auteur)

International audience; The chemical composition of freezing extenders plays a major role in sperm cell survival during cryopreservation. In this study we compared two extenders for freezing stallion semen: INRA82 extender (as a control) versus INRA96 (R) extender, both supplemented with egg yolk and glycerol. INRA82 contains milk, whereas INRA96 (R) is a chemically- defined extender developed for fresh semen storage at 4 degrees C or 15 degrees C. Semen from 3 stallions (7 ejaculates per stallion) was frozen in both extenders. In vitro analyses of post-thaw motility of sperm cells (computer-assisted analysis) and of membrane integrity (flow cytometry analysis) were performed. Then a fertility trial was conducted. Inseminations were conducted in a total of 84 mare cycles. INRA96 (R) extender supplemented with egg yolk and glycerol significantly improved per-cycle pregnancy rates compared with INRA82 (71% versus 40%, p < 0.01). In agreement with these fertility results, membrane integrity was better preserved in INRA96 (R) than in INRA82. In contrast, motility parameters were significantly higher in INRA82 than in INRA96 (R). Further research is needed to understand how INRA96 (R) components protect sperm cells during the cryopreservation process and highly increase their fertility potential compared with INRA82 components.

La congelation du sperme de salmonides a fait l'objet de nombreuses etudes rapportees dans les syntheses bibliographiques realisees par Horton et Ott (1976), Scott et Baynes (1980), Stoss (1983) et plus recemment par Leung et Jamieson (1991). La plupart des auteurs s'accordent sur la tres grande variabilite des resultats obtenus avec le sperme de salmonides congele. Les dilueurs de congelation contiennent souvent des sucres a des concentrations variables : le saccharose est employe a 125 mM chez le saumon atlantique, Salmo salar, (Mounib, 1978) et chez la truite arc-en-ciel, Oncorhynchus mykiss, (Legendre et Billard, 1980) et a 600 mM chez la truite arc-enciel (Holtz et al., 1991); le glucose est utilise a une concentration de 300 mM chez le saumon atlantique (Stoss et Refstie, 1983), chez la truite commune, Salmo trutta, et l'omble chevalier, Salvelinus alpinus, (Piironen, 1993) et 600 mM chez la truite arc-enciel (Holtz et al., 1991). Cependant il n'existe pas d'etude comparative entre les differents glucides ni d'essai systematique sur d'autres sucres alors qu'ils presentent un grand interet en cryoconservation. En effet, Anchordoguy et al. (1987) ont montre que le saccharose et le trehalose interagissent avec les phospholipides de la membrane et augmentent la stabilite de cette derniere pendant la congelation et la decongelation, et concluent que les disaccharides pourraient etre de meilleurs cryoprotecteurs que le glycerol et le dimethylsufoxyde. Dans cette etude nous avons voulu comparer l'effet cryoprotecteur de differents sucres en remplacement du saccharose dans le dilueur de Mounib (1978).

Short-term storage over several days as well as cryopreservation of turbot (Scophthalmus maximus) sperm were studied. Two extenders, Ringer 200 and artificial seminal liquid (ASL2), are suggested for semen collection in order to avoid the deleterious effect of urine contamination, and for the purpose of short-term storage between 0 and 15 degrees C. Oxygen atmosphere is not suitable for turbot sperm storage. Turbot spermatozoa undergo cryopreservation with a high rate of success especially in a sucrose solution with 10% dimethyl sulfoxide (DMSO) and 10% egg yolk.

Un lot de 40 mâles d'une souche de truite arc-en-ciel (Salmo gairdneri) à reproduction printanière a été suivi. Seuls les spermes d'un volume supérieur ou égal à 6 ml ont été caractérisés et congelés en boulettes. L'analyse factorielle des correspondances entre les caractéristiques et la fécondance du sperme après 5 à 9 mois de congélation a montré en particulier que l'aptitude à la cryoconservation peut être caractérisée par l'analyse du plasma séminal : les spermes médiocres sont associés à une osmolarité plasmatique élevée (260 m-osmoles) et/ou une teneur élevée en protéine 42 KD, l'un des constituants majeur de la membrane des spermatozoïdes; les très bons spermes sont associés à l'absence de protéine 42 KD dans le liquide séminal. Ces résultats renforcent l'idée que l'aptitude à la cryoconservation du sperme de truite dépend en premier lieu de la qualité de la membrane des spermatozoïdes., Milt was obtained from 40 males of a spring-spawning strain of rainbow trout (Salmo gairdneri). Only semen with a volume greater or equal to 6 ml were characterized and frozen in pellets. Factorial analysis of correspondences between the physico-chemical characteristics and the fertility of sperm after 5 to 9 months freezing revealed that the fitness for cryopreservation can be particularly characterized by the composition of the seminal plasma: sperm with the lowest capacity to fertilize were associated with a plasma osmolality greater than 260 m-Osm and/or a high level of 42 KD protein, one of the major components of the spermatozoan membrane; sperm with the highest fertilizing ability lacked 42 KD protein in the seminal plasma. These results support the idea that the fitness for cryopreservation of trout sperm primarily depends on the condition of the spermatozoan membrane.

International audience; Cryopreservation trials on rainbow trout (Salmo gairdneri) sperm were carried out using two basic extenders : Mounib's medium (M) and Ménézo's medium (Me) to which were added bovine serum albumin (BSA) and tellurite egg yolk (Institut Pasteur). After 10 p. 100 of DMSO was added to these different deep-freeze diluents (DD), they were mixed with the sperm and then deep-frozen into 100 !Ll pellets on dry ice. The pellets were stored in liquid nitrogen for periods lasting from 3 days to 6 months. The intensity of sperm motility and fertilizing ability were measured before and after cryopreservation. After the sperm was diluted in Ménézo's medium, slight spermatozoon motility was noticed, which probably caused their early exhaustion and would explain the lower fertilizing ability observed after thawing. Mounib's medium gave better results, especially after 10 p. 100 of egg yolk was added. The optimal deep-freeze conditions were : 1/3 dilution, no equilibration after dilution but immediate deep-freezing at a rate of 10 to 40 °C¡min. Thawing had to be carried out rapidly in 10 sec. However, the spermatozoa were altered during the freezing-thawing process, and during insemination more frozen spermatozoa had to be used to equal the fertilization rate obtained with non-frozen sperm. However, the fertile spermatozoa gave normal embryogenesis and no abnormal development was seen up to the vesicle resorption stage. At the end of spermiation, sperm fitness for deep-freezing decreased, perhaps due to sperm senescence. Pooling the sperm of several males partially compensated for the loss of fertilizing ability seen at the end of the reproductive period.

Several methods to control gamete availability in northern pike were investigated. Precocious induction of ovulation was achieved with a single injection of partially purified salmon gonadotropin, but the treatment should be administered soon after capture in order to avoid ovarian atresia. Ovulated oocytes should be inseminated within 24 hours after ovulation. Loss of fertility due to aging occurred 1-3 days after ovulation. High doses of progesterone induced a threefold increase of sperm release. Cryopreservation of diluted sperm at -196 C led to variable fertilizing capacities after thawing, depending on the donor male. A diluent originally tested for trout was used successfully for artificial insemination.