Evaluation by re-derivation of a paternal line after 18 generations on seminal traits, proteome and fertility

[EN] Males from a paternal line selected for growth traits were used to produce semen doses at insemination centres and farms in a breeding scheme for rabbit meat production. The aim of this study was to assess whether a program of selection by daily gain in fattening period changed the seminal traits, plasma and sperm proteome and the fertility of semen when used in artificial insemination. Thirty-nine males from a paternal line were obtained by re-derivation from vitrified embryos with a difference of 18 generations (G21V and G39V). Sperm production parameters, morphological traits, sperm motility parameters and viability were evaluated from ejaculates. Seminal plasma and sperm proteome of three pool ejaculates from 10 mature males of each group were analysed and semen doses were used to inseminate 311 females. Only the percentage of abnormal sperm showed significant differences, with G21V presenting fewer abnormal sperm than G39V (10.5 +/- 2.63 vs 23.8 +/- 1.98). The discriminant analysis (DA-PLS) showed a clear effect of the generation for plasma and sperm proteome. In seminal plasma, 643 proteins were reported and 64 proteins were differentially expressed, of which 56 were overexpressed in G39V (87.5%). Sperm proteome reported 1360 proteins with 132 differentially abundant proteins. Of the total, 89 proteins were overexpressed in G39V (67.4%). From the 64 and 132 differentially abundant proteins of plasma and sperm, 19 and 26 had a FC >1.5, 12 and 13 of them belonging to the Oryctolagus cuniculus taxonomy, respectively. Despite observing differences in important proteins related to capacitation, sperm motility or immunoprotection and consequently to the fertilization process (TMPRSS2, Serpin family, Farn71f1, ATPase H+ transporting accessory protein 2, carbonic anhydrase 2, UDP-glucose glycoprotein glucosyltransferase 2), no differences in fertility and prolificacy were detected when commercial seminal doses were used for insemination from both male groups. However, overabundance of KIAA1324 protein can be related to the increase in abnormal sperm after selection by growth rate.This research was supported by AGL2017-85162-C2-1-R research project funded by Ministerio de Economia, Industria y Competitividad (MICINN, Spain). X Garcia-Dominguez was supported by a research grant from MICINN (BES-2015-072429). English text version was revised by N. Macowan English Language Service.Juárez, JD.; Marco-Jiménez, F.; Talaván, AM.; García-Domínguez, X.; Viudes-De-Castro, MP.; Lavara, R.; Vicente Antón, JS. 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Extra-uterine (abdominal) full term foetus in a 15-day pregnant rabbit

[EN] Background: While ectopic pregnancies account for 1-2% of all pregnancies, abdominal pregnancy is extremely rare, accounting for approximately 1% of ectopic pregnancies. Extrauterine abdominal pregnancy is defined as the implantation and development of an embryo in the peritoneal cavity. The present report is the first of an incidental case of abdominal pregnancy within four full-term foetus simultaneously with 2 weeks of physiological gestation in a healthy doe rabbit. Case presentation: The doe was born on November 3, 2014 and the first partum took place on May 18, 2015. The doe had previously delivered and weaned an average of 12.0 +/- 1.41 live kits at birth (no stillbirths were recorded) during 5 consecutive pregnancies. The last mating was on December 18, 2015 and the detection of pregnancy failure post breeding (by abdominal palpation) on December 31, 2015. Then, the doe was artificially inseminated on January 27, 2016, diagnosed pregnant on February 11, 2016 and subsequently euthanized to recover the foetus. A ventral midline incision revealed a reproductive tract with 12 implantation sites with 15 days old foetus and 4 term foetus in abdominal cavity. There were two foetus floating on either side of the abdominal cavity and two suspended near the greater curvature of the stomach. They were attached to internal organs by means of one or 2 thread-like blood vessels that linked them to the abdominal surfaces. Conclusions: In our opinion a systematic monitoring of rabbit breeding should be included to fully understand and enhance current knowledge of this phenomenon of abdominal pregnancy.This work was supported by Spanish Research Project AGL2014-53405-C2-1-P (Interministerial Commission on Science and Technology).Marco-Jiménez, F.; Garcia-Dominguez, X.; Valdes-Hernández, J.; Vicente Antón, JS. (2017). 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Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (degrees Cmin(-1)) from -6 degrees C to -100 degrees C using a programmable freezer: slow (-15 degrees Cmin(-1), S), medium (-40 degrees Cmin(-1), M) or fast (-60 degrees Cmin(-1), F). After thawing (50 degrees C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15 degrees Cmin(-1)) and fast (-60 degrees Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability

Use of powdered egg yolk vs fresh egg yolk for the cryopreservation of ovine semen

Egg yolk is a common additive to sperm cryopreservation diluents. Because of its animal origin, however, it also represents a potential risk of microbiological contamination in the diluent. This potential contamination can be avoided by using powdered egg yolk, instead of fresh egg yolk, as it is pasteurized. This study was conducted to determine ram sperm cryosurvival was affected by the type of egg yolk used (powdered egg yolk or fresh egg yolk) and by yolk concentration (10, 15 or 20%) in the diluent. Microbiological analyses were also performed to quantify the microbiological contamination in the diluents containing the two types of egg yolk. Sperm cryosurvival was determined by motility and morphology analyses after thawing. Motility parameters were assessed using a computer-assisted sperm analysis (CASA) system, and the percentage of sperm with a normal apical ridge was evaluated using a differential interference contrast microscope. No significant differences were observed between diluents in the percentage of sperm with normal apical ridge. However, higher percentages of total motile cells were observed for samples containing powdered egg yolk (69%) compared to samples containing fresh egg yolk (60%). However, sperm in diluents containing fresh egg yolk, exhibited higher values for average-path velocity, straight-line velocity and beat cross frequency and lower values for amplitude of lateral head displacement (p < 0.05), compared to cells in diluents containing powdered egg yolk. Microbiological contamination was similar (< 200 CFU/ml) in both diluents, and no bacterial growth was observed in either, when antibiotics were added. Therefore, powdered egg yolk can be effective used in diluents for the freezing of ram semen. However, the in vivo fertility of sperm frozen in diluents containing powdered egg yolk should be tested, as some motility parameters were different for sperm treated with powdered egg yolk compared to fresh egg yolk

Oocyte quality and in vivo embryo survival after ovarian stimulation in nulliparous and multiparous rabbit does

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viabilit

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer-assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time-consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean standard error length, of 2.57 +/- 0.01 mu m vs 2.54 +/- 0.02 mu m, width of 2.22 +/- 0.02 mu m vs 2.26 +/- 0.04 mu m, surface area of 4.44 +/- 0.02 mu m(2) vs 4.50 +/- 0.04 mu m(2) and perimeter of 7.70 +/- 0.02 mu m vs 7.73 +/- 0.04 mu m using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa, were found to be smaller than those of sharpsnout sea bream,,spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 +/- 0.01 mu m vs 1.94 +/- 0.02 mu m, head width of 1.80 +/- 0.01 mu m vs 1.78 +/- 0.02 mu m, surface area of 3.16 +/- 0.03 mu m(2) vs 3.18 +/- 0.06 mu m(2) and perimeter of 6.52 +/- 0.04 mu m vs 6.56 0.08 mu m using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results