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Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C. The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer. Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P.05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2. Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96. These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells.

Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in adult cyclic jennies and in vitro matured in tissue culture medium 199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. They were preincubated with oviductal fluid for 30 minutes, coincubated with frozen-thawed donkey semen treated with procaine for 18 hours, and cultured for 30 hours in Dulbecco's Modified Eagle Medium-F12 supplemented with NaHCO3, fetal calf serum, and gentamycin. From the five OPU sessions, we collected 92 COCs in 193 follicles (48%) with an average of 4.2 COCs per jenny. All COCs were expanded after more than 24-hour IVM. At collection, jennies oocytes contained a germinal vesicle. Metaphase 1 oocytes were observed after 30-hour IVM and 44% were in metaphase 2 after 34-hour IVM. In our conditions, IVM of donkey oocytes was slower than IVM of equine oocytes and optimal duration for donkey oocytes IVM may be 34 hours. Only 15% of jennies oocytes contained two pronuclei after coincubation with donkey spermatozoa and none of them developed further after 48 hours post-IVF. Moreover, some parthenogenetic activation occurred. Thus, the treatment of donkey sperm with procaine may not be efficient for IVF. In conclusion, we established for the first time conditions for OPU in jennies with high recovery rates. We reported that IVM of jennies oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture and we described for the first time the chronology of IVM of donkey oocytes. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure were evaluated. Use of the first three jets of ejaculate before the centrifugation did not improve post-thaw motility compared to use of the whole semen (25% versus 25%, 2 stallions x 12 ejaculates, P0.80). When the first dilution was performed in E2 at 22 degrees C instead of in E1 at 37 degrees C, motility was slightly improved (38% versus 36%, n283 ejaculates per group, P0.04) but fertility was similar (51% versus 58%, n196 cycles per group, P0.10). Coating the spermatozoa with 0.5, 1, 2, 4 and 8mM of Concanavalin A resulted in unchanged post-thaw motility (6 stallions x 3 ejaculates, P0.05). The extender E2 was modified or supplemented with different substances. Increasing egg yolk concentration from 2 to 4% (v/v) did not increase post-thaw motility (42% versus 34%, 6 stallions x 2 ejaculates, P0.05). Different glycerol concentrations (range: 1.7-3.7%) had no significant effect on post-thaw motility even though 2.4-2.8% resulted in a nonsignificant higher motility (7 stallions x 2 ejaculates, P0.05). Glutamine at 50mM in E2 improved post-thaw motility compared with no glutamine (49% versus 46%, n584 ejaculates per group, P0.0001) but not fertility (53% versus 54%, n451 cycles per group, P0.80). Thawing at 75 degrees C for 10s slightly increased motility after 120 min at 37 degrees C (6 stallions x 1 ejaculate, P0.05) but no effect on per-cycle fertility was noted (32% (19 cycles) versus 41% (17 cycles), P0.50). When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was unaffected compared with dilution in one step (36% versus 38%, 6 stallions x 1 ejaculate, P0.20).

International audience; The ovarian physiology of the mare is an interesting model. Actually, we could highlight that in contrast with other domestic mammals, no preovulatory LH surge occurs in this species, but LH rises gradually during oestrus with a maximum 1–2 days after ovulation. Moreover, in vitro maturation rate of oocytes remains low in the mare, and in vitro fertilisation methods give only limited success. Thus, follicular development and maturation could involve, at least in part, some species-specific mechanisms and studies on follicular fluid composition may contribute to understanding these events, as well as to alleviate some of the problems encountered. The studies reported here are on the evolution of intrafollicular content of steroids, protein patterns and insulin-like growth factor binding proteins (IGFBPs) profiles during follicular growth, maturation and atresia in the mare, in relation to follicular and oocyte quality. We demonstrated that (1) follicular growth of the dominant follicle is associated with an increase in intrafollicular oestradiol-17β and progesterone levels and a decrease in IGFBP-2 and IGFBP-5, (2) follicular maturation is associated with a decrease in intrafollicular estradiol-17β level, a further increase in progesterone level, a slight increase in IGFBP-2 and finally, the induction of the follicular expression of an Mr 200 000 protein, whose identity is still unknown, and (3) follicular preovulatory regression and atresia are characterised by alterations in steroidogenesis, as well as an increase in intrafollicular levels of IGFBP-2, IGFBP-4 and IGFBP-5. Interestingly, we obtained data which suggest that the expression of the Mr 200 000 protein relates to oocyte resumption of meiosis and cumulus expansion. Finally, relationships between follicular content and follicular quality needs further work in the mare, in order to predict fertility and to increase the embryo production in vivo and in vitro in this species.; Chez la jument, le taux de LH ne présente pas de pic au stade préovulatoire, mais plutôt une augmentation progressive présentant un maximum le lendemain de l’ovulation. D’autre part, le taux de maturation in vitro des ovocytes équins est faible, et la fécondation in vitro ne présente qu’un taux de succès limité. La croissance et la maturation folliculaire pourraient mettre en jeu des mécanismes spécifiques de l’espèce, et l’étude de la composition du fluide folliculaire permettra peut-être de mieux comprendre ces événements. Dans ce travail, l’évolution du contenu intrafolliculaire en stéroı̈des, protéines et IGFBP a été étudiée au cours de la croissance, la maturation et l’atrésie folliculaire chez la jument. Elle a été mise en relation avec la qualité folliculaire et ovocytaire. Nous avons montré que: (1) la croissance du follicule dominant se caractérise par l’augmentation des taux intrafolliculaires d’estradiol-17β et de progestérone, et la chute des taux d’IGFBP-2 et d’IGFBP-5, (2) la maturation du follicule dominant s’accompagne de la baisse du taux d’estradiol-17β, l’augmentation du taux de progestérone et d’IGFBP-2, ainsi que par l’expression d’une protéine de Mr 200 000 (p200) dont l’identité est inconnue, et enfin, (3) la régression préovulatoire et l’atrésie folliculaire se caractérisent par l’altération de la stéroidogenèse et l’augmentation des taux d’IGFBP-2, IGFBP-4 et IGFBP-5. Nos résultats montrent que l’expression de la p200 est liée à la maturation ovocytaire et à l’expansion du cumulus.

The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline.

The high-molecular-weight proteins of equine follicular fluid were examined to determine whether some polypeptides are unique to certain physiological conditions. Fluids from ovarian follicles of various diameters and physiological stages during the follicular phase were recovered by ultrasound-guided follicular aspiration. Granulosa cells and cumulus-oocyte complexes (COC) were recovered by scraping the intrafollicular wall during puncture. Follicular fluids and corresponding serum, as well as granulosa cell lysates, were analyzed by one-dimensional SDS-PAGE and silver staining. COC morphology was assessed microscopically. A 200-kDa protein band was demonstrated in fluids from preovulatory follicles, in natural conditions or after induction of ovulation. This protein band was absent in fluids from follicles at earlier stages, subordinate follicles, and serum. The presence of this protein at the preovulatory (PO) stage was ascertained through recovery of the fluid from follicles twice during their growth. Its appearance was time dependent after induction of ovulation but was not induced by an intrafollicular injection of a physiological dose of progesterone. We also demonstrated the presence of this 200-kDa protein in granulosa cells lysates recovered from preovulatory follicles. The expression of this protein in the follicular fluid was related to the cumulus aspect and chromatin configuration of the enclosed COC. No relation was found between its presence in the follicular fluid at the PO stage and subsequent ovulation of the punctured follicle or embryo production. The identification of this molecule is approached and discussed. These results show a novel PO stage-related protein in equine follicular fluid, which may be involved in the differentiation and maturation mechanisms occurring in the follicle during the preovulatory period.

International audience

Hypo- and hyperthyroidism alter testicular functions in the young. Among T3 receptors, TRalpha1 is ubiquitous, and its previously described knockout leads to an increase in testis weight and sperm production. We tested, for the first time, the hypothesis that TRalpha1-dependent regulation of Sertoli cell (SC) proliferation was directly regulated by TRalpha1 present in these cells. Thus, after crossing with the AMH-Cre line, we generated and analyzed a new line that expressed a dominant-negative TRalpha1 isoform (TRalphaAMI) in SCs only. So-called TRalphaAMI-SC (TRalphaAMI/+ Cre+) mice exhibited similar phenotypic features to the knockout line: heavier testicular weight and higher sperm reserve, in comparison with their adequate controls (TRalphaAMI/+ Cre−). SC density increased significantly as a result of a higher proliferative index at ages Postnatal Day (P) 0 and P3. When explants of control testes were cultured (at age P3), a significant decrease in the proliferation of SCs was observed in re...

Hypo- and hyperthyroidism alter testicular functions in the young. Among T3 receptors, TRalpha1 is ubiquitous, and its previously described knockout leads to an increase in testis weight and sperm production. We tested, for the first time, the hypothesis that TRalpha1-dependent regulation of Sertoli cell (SC) proliferation was directly regulated by TRalpha1 present in these cells. Thus, after crossing with the AMH-Cre line, we generated and analyzed a new line that expressed a dominant-negative TRalpha1 isoform (TRalpha(AMI)) in SCs only. So-called TRalpha(AMI)-SC (TRalpha(AMI/+) Cre(+)) mice exhibited similar phenotypic features to the knockout line: heavier testicular weight and higher sperm reserve, in comparison with their adequate controls (TRalpha(AMI/+) Cre(-)). SC density increased significantly as a result of a higher proliferative index at ages Postnatal Day (P) 0 and P3. When explants of control testes were cultured (at age P3), a significant decrease in the proliferation of SCs was observed in response to an excess of T3. This response was not observed in the TRalpha(AMI)-SC and knockout lines. Finally, when TRalpha(AMI) is present in SCs, the phenotype observed is similar to that of the knockout line. This study demonstrates that T3 limits postnatal SC proliferation by activation of TRalpha1 present in these cells. Moreover, quantitative RT-PCR provided evidence that regulation of the Cdk4/JunD/c-myc pathway was involved in this negative control.

This study tested whether variable temperatures (from -0.5 to 15 degrees 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 degrees C with air vs. 15 degrees 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 degrees C, 17 degrees C, 27 degrees C, 39 degrees C with semen at -0.5 degrees C to 3 degrees C, 4 degrees C to 7 degrees C, 8 degrees C to 10 degrees C, 12 degrees C to 15 degrees C, respectively). Per cycle pregnancy rate (PC) was measured after one artificial insemination (AI) in uterine body of 200 X 10(6) 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 X 8 cycles). Only velocity VCL and ALH were slightly higher at 15 degrees C. In Experiment 2, PC was reduced when ambient temperature was low (semen at -0.5 degrees C to 3 degrees C; PC = 25%) rather than intermediate (semen at 4 degrees C to 7 degrees C; PC = 53%) or high (semen at 8 degrees C to 10 degrees C; PC = 50%) (4 stallions X 8 cycles) (P = 0.002). Sperm stored at -0.5 degrees C to 3 degrees 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 degrees 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 degrees C are detrimental for fertility.

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

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La plupart des espèces d’ânes sauvages et domestiques est en danger d’extinction. Leur préservation nécessite la cryoconservation du sperme, des ovocytes et des embryons. L’objectif de cette étude était de mettre au point une technique de collecte in vivo et de maturation in vitro des ovocytes d’ânesses. Les ovocytes ont été collectés par ponction folliculaire transvaginale sous guide échographique, placés dans un milieu de maturation in vitro pendant 24, 30, 34 ou 38 heures puis analysés. Quatre-vingt-douze ovocytes ont été collectés dans 193 follicules (48%) avec une moyenne de 4,2 ovocytes par ânesse. Après 34 heures de maturation in vitro, 45% des ovocytes ont été matures. Pour la première fois une technique de collecte in vivo et de maturation in vitro d’ovocytes d’ânesses a été développée. Cette technique permet d’envisager la cryoconservation et la fécondation in vitro de ces ovocytes.

International audience; To fertilize the oocyte, the sperm undergoes physiological and biochemical modifications known as “sperm capacitation”. The aim of this study was to analyze by flow cytometry and immunofluorescence analysis the changes induced by several molecules on stallion sperm viability, plasma membrane fluidity, cholesterol content, calcium influx and tyrosine phosphorylation. Moreover, the modifications induced on glycocalyx sperm sugars were studied by means of lectin-based cell microarray analysis. Frozen sperm samples were incubated with calcium ionophore, MβCD, α-L-fucosidase, neurotensin, HSPA8, cAMP and 4-AP. Flow cytometry analysis showed that membrane fluidity was affected by MβCD, cAMP, neurotensin, HSPA8 and 4-AP; cholesterol depletion was induced by MβCD; an increase of intracellular calcium was produced by calcium ionophore and cAMP; and sperm viability was affected by MβCD. Tyrosine phosphorylation increased when sperm was incubated with MβCD and cAMP. Sperm microarray analysis revealed that i) MβCD and cAMP treatment increased α2,3-linked sialic acid and T antigen, whereas decreased α1,3-linked fucose signals; ii) cAMP alone increased α1,6- and α1,2-linked fucoses; iii) MβCD alone reduced sialylated T antigen, lactosamine, αGal, GalNAc, terminating glycans as well as α 1,6fucose in high-mannose glycans. The combination of MβCD + cAMP treatment preserved the α2,3-linked sialic acid increasing, determined the GalNAc, GlcNAc, α 1,2-linked fucose enhancement, whereas reduced T antigen and high-mannose glycans. Our results determine the specific effect of each molecule in order to establish a combination of several agents to achieve an efficient in vitro capacitation technique for stallion sperm.

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