Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

Tumor necrosis factor-α is transferred to equine neonates via colostrum but is not associated with their health status.

We followed the hypothesis that equine neonates with reduced transfer of tumor necrosis factor-α (TNFα) are at increased risk of neonatal infection. We investigated TNFα concentrations in colostrum of healthy mares and blood of their neonates in a non-hospitalized population of Warmblood mares where delivery, neonatal adaptation and health was closely monitored by veterinarians. Concentration of TNFα and IgG was determined in colostrum respective milk and in neonatal blood collected immediately after delivery and 18 h thereafter in 97 foals that were assigned to groups failure of passive transfer (FPT; n = 31) and control (CON; n = 66) based on serum IgG concentration at 18 h of age. Foal health was assessed repeatedly during the first 24 h of life. Statistical analysis was done with p < 0.05 indicating significance. There were no significant differences between foal groups FPT and CON regarding age and parity of dams, gestation length (FPT 343 ± 10, CON 340 ± 8 days) and foal sex. Concentrations of TNFα in colostrum at birth and in foals at 18 h varied but did not differ between groups (colostrum FPT 6.1 ± 9.1, CON 9.9 ± 31.5 ng/ml; foal FPT 2.3 ± 5.9, CON 2.4 ± 5.3 ng/ml; n.s.). There was an increase in the mean serum TNFα concentration until 18 h in foals (n.s. between groups). Results of the present study confirm previous findings of TNFα transfer from the mare to the neonate via colostrum but do not suggest that transfer of TNFα via colostrum is important for protection of the neonate against infectious diseases.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

CONTEXT: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. AIMS: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. METHODS: Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4µg/day for 4weeks followed by 8µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. KEY RESULTS: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. CONCLUSIONS: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. IMPLICATIONS: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Protamine 2 and phospholipase C zeta 1 are possible biomarkers for the diagnosis of male subfertility in frozen-thawed stallion semen.

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = -0.495), PRM2/ZPBP (r = -0.645) and CRISP3/ACR (r = -0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.

Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses.

High progesterone concentrations in the early luteal phase support pregnancy, whereas subphysiological progesterone concentrations delay embryonic development at least until placentation. In this study, fetal growth and development of pregnancy was investigated in pregnancies with prostaglandin F2α-induced low progesterone concentrations (PGF) in the early luteal phase and control pregnancies (CON) in the same mares (n = 12). Mares were inseminated and in PGF pregnancies received the prostaglandin F2α analogue cloprostenol (62.5 µg) on days 0-3 after ovulation to induce subphysiological progesterone concentrations; CON pregnancies remained untreated. Mares were assigned to PGF or CON treatments in alternating order and received the opposite treatment in the following year. Blood was collected and conceptus size determined repeatedly by transrectal (≤day 101) and transabdominal (>day 101) ultrasonography. After birth, foals were weighed, measured and submitted to a clinical examination. Treatment PGF resulted in fewer pregnancies than CON treatment. All foals born from CON pregnancies were healthy and mature, whereas 4/7 PGF pregnancies were either lost (one embryonic death, one abortion) or resulted in the birth of compromised foals (P = 0.018). Size of the conceptus (e.g., diameter day 49: PGF 6.6 ± 0.7, CON 7.7 ± 0.7 cm, P = 0.006) and embryo proper (e.g., crown rump length day 54; PGF 4.4 ± 0.8, CON 5.8 ± 0.6 cm, P = 0.015) differed between treatments. These size differences decreased over time and at birth PGF foals did not differ significantly from CON foals. In conclusion, reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.

Abundance of Anti-Muellerian hormone in cat ovaries and correlation of its plasma concentration with animal age, weight and stage of the estrous cycle.

In female animals of different species, Anti-Müllerian hormone (AMH) is produced by follicular granulosa cells and has been associated with the ovarian follicle pool. Because concentration of AMH in plasma of ovary-intact female cats is apparently more variable than previously assumed, we have analysed AMH concentration in blood of cats (n = 93) presented for routine ovariectomy and assessed ovarian histology and AMH protein expression in the surgically removed ovaries. We hypothesised that AMH is synthesized only in preantral and small antral follicles and that plasma AMH concentration reflects the antral follicle count (AFC). Corpora lutea were detected in 35% of the female cats, whereas plasma progesterone concentration was ≥1 ng/mL in 57% of the cats. Follicular cysts were present in 15 cats (16%). Positive immunostaining for AMH protein was detected in close to all primordial and antral follicles, ovarian cysts, 70% of corpora lutea and 28% of atretic follicles. Concentration of AMH in plasma averaged 6.8 ± 0.5 ng/mL (range 1.3-21.7 ng/mL). The AFC increased with increasing AMH concentration with a moderate positive correlation between AFC and AMH (r = 0.286, p < 0.01). Plasma AMH concentration was not affected by season or cats' age, weight, stage of the estrous cycle and presence of follicular cysts. In conclusion, AMH protein is expressed in all endocrine structures of the cat ovary. While AMH is a marker for the presence of ovarian tissue, its usefulness to assess ovarian function in individual female cats is of limited value.

Shipping duration and temperature influence the characteristics of cryopreserved horse semen stored in different shipping devices for up to 14 days.

This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ -150 °C) or on dry ice (≤ -78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intact sperm (SYBR14/PI), high mitochondrial membrane potential (HMMP; JC1) and DNA fragmentation. Temperature inside the containers was monitored continuously. Until d7, no changes were observed in TMot, PMot, and membrane-intact spermatozoa. Thereafter, all three parameters decreased in semen stored on dry ice but not in a dry shipper (time p < 0.001, time x shipping device p < 0.001). The HMMP decreased continuously over time in both containers with a more pronounced decrease on dry ice compared to the dry shipper (shipping device p < 0.01, time p < 0.001, time x device p < 0.001). The DNA fragmentation increased on d10-14 on dry ice and d14 in the dry shipper (time p < 0.001, time x device p < 0.01). In conclusion, frozen horse semen can be safely stored for up to 7 days on dry ice. Sperm DNA integrity and HMMP, however, were adversely affected after 14 days in both shipping devices.

Psychological stress induces an increase in cholinergic enteric neuromuscular pathways mediated by glucocorticoid receptors.

Introduction: Repeated acute stress (RASt) is known to be associated with gastrointestinal dysfunctions. However, the mechanisms underlying these effects have not yet been fully understood. While glucocorticoids are clearly identified as stress hormones, their involvement in RASt-induced gut dysfunctions remains unclear, as does the function of glucocorticoid receptors (GR). The aim of our study was to evaluate the involvement of GR on RASt-induced changes in gut motility, particularly through the enteric nervous system (ENS). Methods: Using a murine water avoidance stress (WAS) model, we characterized the impact of RASt upon the ENS phenotype and colonic motility. We then evaluated the expression of glucocorticoid receptors in the ENS and their functional impact upon RASt-induced changes in ENS phenotype and motor response. Results: We showed that GR were expressed in myenteric neurons in the distal colon under basal conditions, and that RASt enhanced their nuclear translocation. RASt increased the proportion of ChAT-immunoreactive neurons, the tissue concentration of acetylcholine and enhanced cholinergic neuromuscular transmission as compared to controls. Finally, we showed that a GR-specific antagonist (CORT108297) prevented the increase of acetylcholine colonic tissue level and in vivo colonic motility. Discussion: Our study suggests that RASt-induced functional changes in motility are, at least partly, due to a GR-dependent enhanced cholinergic component in the ENS.

Selection of frozen-thawed stallion semen by microfluidic technology.

The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 µl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 µl) for stallion semen. Frozen-thawed semen from 15 stallions that were previously classified as being of 'good fertility' (GF; n = 8; pregnancy rate ≥ 40%) and 'poor fertility' (PF; n = 7; pregnancy rate < 20%), respectively, was used. Each ejaculate was assessed before and after microfluid recovery for kinetics (CASA), membrane integrity (MI) (SYBR14/PI), membrane functionality (MF) (HOS test), acrosome integrity (Spermac Stain Kit), morphology (Spermac stain kit), mitochondrial membrane potential (MMP) (JC-1) and chromatin integrity (aniline blue staining). Sperm concentration was reduced after sperm recovery in both groups, but more markedly in frozen-thawed semen of PF stallions (p < .05). Microfluid recovery increased total motility, MI, MF and MMP. While there was a significant increase in the percentage of progressively motile sperm after sperm microfluid recovery, there was a decrease in DAP, DSL, VAP, VSL, LIN, WOB and ALH (p < .05). A slight increase (p < .05) was detected in beat-cross frequency. The present results suggest that the ZyMot Multi (850 µl) device selects a specific sperm population from any stallion ejaculate with motile sperm and could therefore be a valid tool for in vitro testing with the aim to predict the fertility of frozen-thawed stallion semen.

Low plasma progesterone concentration during the early luteal phase delays endometrial development and the beginning of placentation in mares.

While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to treatment (TREAT) or control (CON) during two pregnancies. In TREAT pregnancies, mares received a PGF2α analogue for four consecutive days starting on the day of ovulation with the aim to reduce progesterone secretion. Mares were left untreated in CON pregnancies and thus served as their own controls. Endometrial biopsies for analysis of histomorphology, epidermal growth factor (EGF) and EGF receptor (EGFR) mRNA and protein expression in the endometrium, AC, and CG as well as abundance of regulatory T lymphocytes (Tregs) were collected on day 34 of pregnancy. Histomorphometric analysis revealed a higher luminal endometrium and a higher CG epithelium in CON compared to TREAT pregnancies. Abundance of mRNA for EGF and EGFR was large in the endometrium, AC and CG but did not differ between TREAT and CON pregnancies. The number of endometrial regulatory T lymphocytes was reduced in TREAT compared to CON pregnancies, adding further aspects to the potentially detrimental effects of reduced progesterone concentrations on equine pregnancy.

Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment.

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 µg/day for 4 weeks followed by 8 µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function.

"Fine feathers make fine birds" - The mammalian sperm plasma membrane lipid composition and effects on assisted reproduction.

The sperm plasma membrane is important in modulating many sperm functions. The sperm membrane is composed of a complex mixture of lipids including phospholipids, glycolipids and sterols. There are differences of sperm membrane composition among mammalian species with two groups differing in the most abundant polyunsaturated fatty acid (PUFA), either docosahexaenoic (ω-3 PUFA) or docosapentaenoic acid (ω-6 PUFA). During testicular and epididymal maturation, composition of the sperm plasma membrane evolves with spermatozoa gaining the capacity for fertilization. The importance of fatty acid metabolism for complete spermatogenesis has been elucidated using gene knockout mice. During epididymal transit cholesterol content decreases and PUFA content increases, conferring more fluidity properties to the sperm membrane. The relatively lesser content of antioxidant enzymes and the relatively larger content of PUFA make the spermatozoa particularly susceptible to lipid peroxidation during sperm preservation. In numerous studies, there was adding of PUFA and antioxidants to the diet of animals or to semen extenders with the aim to improve sperm membrane integrity. This review highlights the current knowledge on the sperm membrane composition and effects on sperm function in mammalian domestic animals.

Deslorelin Slow-Release Implants Delay Ovulation and Increase Plasma AMH Concentration and Small Antral Follicles in Haflinger Mares.

There is an increasing interest in the manipulation of ovarian follicular populations in large domestic animals because this could prove beneficial for assisted reproductive techniques such as ovum pick-up (OPU). The aim of the present study was to evaluate the effects of deslorelin slow-release implants (SRI) on the interovulatory interval, antral follicle count (AFC), number of follicles of different size ranges and plasma anti-Muellerian hormone (AMH) concentration in mares. To synchronize their estrous cycles, Haflinger mares (n = 12) were treated twice with a PGF2α analogue. One day after the second injection (day 0), mares received a 9.4 mg deslorelin SRI (group DES, n = 6) or 1.25 mg deslorelin in a short-acting formulation (CON; n = 6), respectively. Regular transrectal ultrasonography of the genital tract was performed and blood samples were collected for the analysis of progesterone, AMH and gonadotrophins. The interval from implant insertion to the first spontaneous ovulation was 23.8 ± 10.5 days in group DES compared to 17.0 ± 3.9 days in group CON (p < 0.05). For the concentrations of LH, FSH and AMH, interactions between time and treatment were detected (p < 0.05). The AFC and the mean number of follicles with 5 to 10, 10 to 15 and 15 to 20 mm in diameter changed over time (p < 0.05). A time x treatment interaction was demonstrated for follicles of 10 to 15 mm in diameter (p < 0.05). The changes in this follicular subpopulation were reflected by increased plasma AMH concentration in group DES. In conclusion, 9.4 mg deslorelin implants show minor effects with regard to estrus suppression in mares, whereas the changes in the subpopulation of small ovarian follicles could be a promising tool for preparation of mares for OPU.

Diestrous Ovulations in Pregnant Mares as a Response to Low Early Postovulatory Progestogen Concentration.

Spontaneous prolongation of the luteal phase has been described in horses, but the underlying causes are still unclear. The present study investigated details of gonadotrophin and progestogen secretion in pregnant mares (n = 11) with or without experimentally reduced early postovulatory luteal function. From days 0 to 3 after ovulation, they were treated with the prostaglandin F2α (PGF2α) analogue cloprostenol or left untreated. After conceptus collection on day 34, they were assigned to the opposite treatment. Mares were affiliated to the group primary corpus luteum (n = 6) or diestrous corpus luteum (n = 5) depending on diestrous corpus luteum (CL) detection in the PGF pregnancy. For statistical comparisons, a p-value < 0.05 was significant. There was an effect of treatment (p < 0.01), but not of group on progestogen concentration. The concentration of LH was higher in PGF-treated than in untreated pregnancies (p < 0.05), but did not differ between groups. The FSH concentration did not differ between groups nor treatments. The total luteal tissue area was greater in mares with a diestrous ovulation during the PGF treatment pregnancy. Low progestogen concentration in the early postovulatory phase diminish the negative feedback on the hypothalamic-pituitary axis in early pregnancy and, thus, stimulate a luteal tissue response. Detection of secondary CL at the time of pregnancy examination in mares may reflect that early post-ovulatory progestogen concentrations were low.

Stress Response of Beagle Dogs to Repeated Short-Distance Road Transport.

This study aimed to characterize the response of transport-naïve dogs to one and two-hour road transports based on cortisol in saliva and blood plasma, heart rate, heart rate variability (HRV), neutrophil to lymphocyte (N/L) ratio and behavior. Two persons familiar to the dogs were present during transports and control experiments. We hypothesized that transport elicits a stress response, which decreases with repeated transports. Beagle dogs were allocated to three groups (n = 6 each). Group 1 served as control in the stable in week 1 and was transported for one hour in weeks 2, 3 and 4. Groups 2 and 3 served as controls in a non-moving vehicle and in the stable, respectively, in week 2. All three groups were transported for two hours in week 6. Cortisol concentration increased during transports (p < 0.001), and this increase remained constant with repeated transports. Cortisol release during two-hour transports was not affected by transport experience. Cortisol concentration increased twofold in plasma and eightfold in saliva, indicating an increase in free cortisol. The N/L ratio increased during transport (p < 0.05). Heart rate increased at the beginning of transport while HRV decreased (p < 0.001). Heart rate and HRV neither differed among weeks nor between animals with different transport experience. During transports, but also in the stationary vehicle, dogs were mostly sitting, and time spent standing decreased during experiments (p < 0.001). Licking the mouth was the most frequent behavior during transports but not in the stationary vehicle (p < 0.01). In conclusion, a transport-induced stress response was evident in dogs. There was no habituation with repeated transports, and transported dogs may suffer from motion sickness.

Hatching of Globodera pallida Induced by Root Exudates Is Not Influenced by Soil Microbiota Composition.

Plant-parasitic nematodes are among the most harmful pests of cultivated crops causing important economic losses. The ban of chemical nematicides requires the development of alternative agroecological approaches to protect crops against nematodes. For cyst nematodes, egg hatching is stimulated by host plant root exudates. Inducing "suicide hatching" of nematode second-stage juveniles (J2), using root exudates in the absence of the host plant, may constitute an effective and innovative biocontrol method to control cyst nematodes. However, before considering the development of this approach, understanding the effect of soil biotic component on cyst nematode hatching by root exudates is a major issue. The effectiveness of this approach could be modulated by other soil organisms consuming root exudates for growth as soil microbiota, and this must be evaluated. To do that, four different native agricultural soils were selected based on their physicochemical properties and their microbiota composition were characterized by rDNA metabarcoding. To disentangle the effect of microbiota from that of soil on hatching, four recolonized artificial soils were obtained by inoculating a common sterile soil matrix with the microbiota proceeding from each agricultural soil. Each soil was then inoculated with cysts of the potato cyst nematode, Globodera pallida, and low or high doses of potato root exudates (PREs) were applied. After 40 days, viable J2 remaining in cysts were counted to determine the efficiency of root exudates to stimulate hatching in different soils. Results showed that (i) when physicochemical and microbiota compositions varied among native soils, the hatching rates remained very high albeit small differences were measured and no dose effect was detected and (ii) when only microbiota composition varied among recolonized soils, the hatching rates were also high at the highest dose of PREs, but a strong dose effect was highlighted. This study shows that abiotic and biotic factors may not compromise the development of methods based on suicide hatching of cyst nematodes, using root exudates, molecules inducing J2 hatch, or trap crops.

Plant-parasite coevolution: A weak signature of local adaptation between Peruvian Globodera pallida populations and wild potatoes.

Plant-parasite coevolution has generated much interest and studies to understand and manage diseases in agriculture. Such a reciprocal evolutionary process could lead to a pattern of local adaptation between plants and parasites. Based on the phylogeography of each partner, the present study tested the hypothesis of local adaptation between the potato cyst nematode Globodera pallida and wild potatoes in Peru. The measured fitness trait was the hatching of cysts which is induced by host root exudates. Using a cross-hatching assay between 13 populations of G. pallida and root exudates from 12 wild potatoes, our results did not show a strong pattern of local adaptation of the parasite but the sympatric combinations induced better hatching of cysts than allopatric combinations, and there was a negative relationship between the hatching percentage and the geographical distance between nematode populations and wild potatoes. Moreover, a strong effect of the geographic origin of root exudates was found, with root exudates from south of Peru inducing better hatching than root exudates from north of Peru. These results could be useful to develop new biocontrol products or potato cultivars to limit damages caused by G. pallida.

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Controlled delay of the expulsive phase of foaling affects sympathoadrenal activity and acid base balance of foals in the immediate postnatal phase.

Stress at foaling has been demonstrated to delay birth. In this study, we followed the hypothesis that even a short delay of foaling increases catecholamine and cortisol release in foals, induces acidosis and impairs neonatal adaptation. Foaling was prolonged for 5 min by transferring mares to an unfamiliar environment at rupture of the allantochorion (group delay, n = 6) while control mares (n = 5) were left undisturbed. In their foals, times from birth to first standing and first suckling, heart rate, heart rate variability (HRV) and salivary cortisol concentration were analysed. Blood for analysis of epinephrine, norepinephrine, hematology and blood gases was collected directly and 30 min after birth. Statistical comparisons were made by repeated measures ANOVA. Times to first standing and suckling did not differ between groups. Fetal heart rate remained unchanged during birth and increased within 15 min postnatum (p < 0.001) while HRV decreased during the first hour of life in foals of both groups (p < 0.05). Immediately after birth, actual base excess was lower in foals with delayed birth than in control foals (p < 0.05). Epinephrine concentration immediately after birth was higher in group delay foals and increased from 0 to 30 min after birth in control foals (time p < 0.001, time x group p = 0.001). Cortisol concentration peaked at 1 h after birth in both groups (p < 0.001). Leukocyte and PMN count decreased from 0 to 30 min after birth (p < 0.001). In conclusion, a 5-min delay at foaling affected epinephrine release and acid base balance, but was without further effect on neonatal adaptation.

Microsatellite markers reveal two genetic groups in European populations of the carrot cyst nematode Heterodera carotae.

The cyst nematode Heterodera carotae, which parasitizes carrot roots, has been recorded in many countries in Europe (Italy, The Netherlands, Switzerland, France, Denmark, …), in South Africa and in North America (Canada, USA). To date, there is a lack of knowledge about the genetic structure of the populations of this economically important nematode. The aim of this work was to study the structuration of the genetic diversity of the carrot cyst nematode at the European scale. We have developed a set of thirteen polymorphic microsatellite markers and used it to genotype seventeen European populations of H. carotae coming from France, Switzerland, Italy, Denmark and one non-European population from Canada. As previously showed for other cyst nematode species, the H. carotae populations were characterised by a strong heterozygote deficit. A Bayesian clustering analysis revealed two distinct genetic clusters, with one group located in the north of Europe and a second one located in the south of Europe. Moreover, our results highlighted rather limited gene flow at small spatial scale and some events of long distance migration. This first investigation of the genetic diversity of H. carotae populations would be useful to develop sustainable control strategies.