J.S. Vicente

Effect of freezing–thawing protocols on the performance of semen from three rabbit lines after artificial insemination

The effect of different freezing and thawing protocols on the results observed after artificial insemination with semen from three different rabbit lines (two maternal lines selected for litter size at weaning, lines A and V, and one line selected for growth rate from weaning to slaughter, line R) was studied. The sperm were frozen with a Tris-citric acid-glucose extender which included 1.75 M DMSO and 0.05 M sucrose as cryoprotectants. The straws were cooled to 5 degrees C for 45 min and then some of them were frozen in a freezer at -30 degrees C for 30 min, whereas the other group of straws were frozen in liquid nitrogen vapor (LNV, 5 cm above the liquid nitrogen level) for 10 min. Straws were thawed at two different temperatures: 50 or 70 degrees C for 10-12s. Significant differences were observed between freezing-thawing protocols, obtaining better results in fertility rate (percentage of pregnant females) when sperm had been frozen in LNV (fertility rate increased between 30 and 50 points in all the lines); the best prolificacy was observed when sperm had been frozen in LNV and thawed at 50 degrees C (70% versus 32% fertility rate, P<0.01 and 7.4 versus 5.9 total number of young born, P<0.01 when sperm had been frozen in LNV or at -30 degrees C and thawed at 50 degrees C, respectively). As for the rabbit line, significant differences were observed between lines in fertility rate (62 and 68% versus 45% fertility rate for lines A, V and R, P<0.01), and total number of young born (5.8 versus 6.9 versus 4.6 total number of young born for lines A, V and R, P=0.02). The best results for all lines in both fertility and total number of young born were observed when sperm had been frozen in LNV and thawed at 50 degrees C (85% versus 84% versus 50% fertility rate and 6.7 versus 8.3 versus 7.3 total number of young born for lines A, V and R, respectively), when compared to the results of the control group, frozen at -30 degrees C and thawed at 50 degrees C (30% versus 52% versus 19% fertility rate and 6.7 versus 6.4 versus 4.5 total number of young born for lines A, V and R, respectively). In conclusion, the best results (fertility rate and prolificacy) for all the rabbit lines were obtained after freezing in liquid nitrogen vapor and thawing at 50 degrees C, being more pronounced in the line selected for high growth rate (line R).

Seminal plasma composition from ejaculates collected by artificial vagina and electroejaculation in Guirra ram.

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10-25 kDa rank, the IMAGE ANALYSIS software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.

Does storage time in LN2 influence survival and pregnancy outcome of vitrified rabbit embryos

Vitrification is one of the most widely used techniques for embryo cryopreservation. The aim of this work was to study the effect of storage time in liquid nitrogen on vitrified rabbit embryos. A total of 1467 vitrified rabbit embryos were transferred into 174 females. The embryos had been maintained in liquid nitrogen during 3 different periods, A) < 1 year (98 transfers, 827 embryos); B) 2-5 years (44 transfers, 360 embryos) and C) > 15 years (32 transfers, 280 embryos). A generalized linear model was used to determine the effect of period on pregnancy and birth rates. A Bayesian approach was applied to analyze the survival rate of the vitrified embryos. In all analyses the number of transferred embryos was included as covariate. It was observed that neither the period of storage nor the number of transferred embryos affected pregnancy rate, and all periods presented similar pregnancy rates (0.85 ± 0.04; 0.86 ± 0.05; 0.78 ± 0.07 for A, B and C). Fertility at birth was affected by the number of transferred embryos, but non-significant differences between periods were detected (0.77 ± 0.04, 0.75 ± 0.07; 0.69 ± 0.08 for A, B and C). Also, the posterior means (highest posterior density intervals at 95%) of embryo survival at birth from pregnant females were similar between the different periods (47 [41 53]; 47 [38 56]; 42 [31 54]; for A, B and C). Results obtained in the present experiment point out that vitrified embryos could be stored in liquid nitrogen during at least fifteen years, achieving good pregnancy rate, fertility and survival at birth.

Effect of an asynchrony between ovulation and insemination on the results obtained after insemination with fresh or frozen sperm in rabbits

The effects of the introduction of an 8-h asynchrony between ovulation and insemination on litter size components from rabbits were assessed. A total of 202 females belonging to a maternal line were used. Fresh and frozen sperm were used to perform the inseminations. Sperm was frozen with an extender composed of 1.75 M DMSO and 0.05 M sucrose. Four experimental groups were obtained depending on the type of sperm used (fresh or frozen) and on the moment that ovulation had been induced relative to the insemination (at the same time as insemination (t(0)) or 8 h before insemination (t(8))). Laparoscopy was performed on 12th day of pregnancy in pregnant females, and the ovulation rate, normal and total implanted embryos were noted. At kindling, total and live-born rabbits were noted. Results showed that better results were obtained after insemination with fresh semen than with frozen sperm (for females in the group t(0): 79% versus 61% fertility rate, 10.2 versus 6.4 normal implanted embryos and 8.1 versus 5.2 total number born, for fresh and frozen sperm, respectively). On the other hand, after the introduction of an 8-h asynchrony between ovulation and insemination, results were lower for both fresh (50% fertility rate, 7.5 normal implanted embryos and 5.7 total number born for the group of the asynchrony) and frozen sperm (31% fertility rate, 4.6 normal implanted embryos and 3.4 total number born for the group of the asynchrony). Although an approach between the moment of insemination and ovulation is justified when sperm survival could be compromised, results observed after the induction of an 8-h asynchrony were not those expected, perhaps due to the ageing of the oocytes before being fertilised, leading to both lack of fertilisation or early embryonic mortality.

A simple method for freezing rabbit semen with successful results on fertility and prolificity

The experiments were carried out to evaluate a simple method of freezing rabbit spermatozoa. The effect of glucose, lactose or sucrose on post-thawing sperm quality was studied. The results showed that addition of sucrose improved motility and acrosomal integrity (57% of progressive motile spermatozoa vs 43% and 42%, and 68% of normal acrosome vs 56 and 57%, sucrose, lactose and glucose, respectively). Then, a Tris-citrate medium with 0.05 M sucrose and 1.75 M dimethylsulphoxide (DMSO) was used to analyse the effect of frozen semen on the reproductive performance of does. Insemination of does with fresh and frozen semen showed no differences in fertility rate and prolificity between fresh and frozen semen (80% and 8.1 young rabbits, respectively).

Effect of Embryonic Genotype on Reference Gene Selection for RT‐qPCR Normalization

To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. In rabbits, there are classic stable reference genes that have been identified for normalization in oocytes and pre-implantation stage embryos. However, effects of embryonic genotype on reference gene selection have not been elucidated. The aim of this study was to test (i) the stability of mRNA transcription level for histone (H2afz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in rabbit blastocysts from two lines selected by different criteria (litter size and post-weaning daily weight gain) and (ii) its influence on biological significance examined by means of a set of embryonic transcripts, such as POU5F1 (Oct-4), epidermal growth factor receptor (erbB3), transforming growth factor-beta2, vascular endothelial growth factor and gamma interferon (Ifn-gamma). The geNorm, NormFinder and BestKeeper programs showed similar results, pointing out that H2afz and GAPDH were the most stable reference genes in rabbits selected on litter size at weaning. Moreover, our study revealed that embryonic genotype affected target gene expression when a single reference gene was used to analyse mRNA expression in blastocysts. Results showed that GAPDH gene is better than H2afz for gene expression studies of both embryo genotypes. A normalization factor derived from H2afz and GAPDH is likely to be appropriate when RT-qPCR was performed in rabbit embryos with different genotypes.

Differential mRNA expression in rabbit in vivo pre-implantatory embryos.

To study genes involved in embryo developmental competence and implantation in rabbits, the expression of a panel of genes related to pluripotency, angiogenesis, proliferation, apoptosis and differentiation were evaluated in late rabbit blastocysts. Thirty nulliparous does were used to obtain a total of 184 in vivo-derived blastocysts on days 4, 5 and 6 of development. The relative transcript abundance of vascular endothelial growth factor (VEGF), epidermal growth factor receptor 3 (erbB3), transforming growth factor β2 (TGF β2) and transcription factor OCT-4 were analysed from eight pools of each stage of development, using quantitative real-time reverse transcriptase-polymerase chain reaction (qrtRT-PCR). mRNA expression was detected for all genes in 4-, 5- and 6-day-old blastocysts, according to blastocyst growth and implantation proximity. Significant differences in OCT-4, VEGF and TGF β2 expression were observed between days of development. Results show a down-regulation of OCT-4 from the 4th day, contrasting with the up-regulation of VEGF and TGF β2 at 6-day-old blastocyst. These findings corroborate the importance of VEGF and TGF β2 in rabbit embryo development and implantation and suggest a possible regulator role of OCT-4 in embryonic angiogenetic factors. On the other hand, no differences were found for erbB3 expression. Therefore, the study of specific gene transcripts in rabbit blastocyst could provide novel embryo developmental competence markers and might be used as a new tool for further studies of embryo quality and in vitro development.

Genetic parameter estimates for semen production traits and growth rate of a paternal rabbit line

Variance components of sperm production traits (volume in ml, V; concentration in ×10⁶ sperm/ml, CN; sperm production in ×10⁶ sperm, PROD) were estimated in a paternal line of rabbit selected for 25 generations based on daily weight gain (DG, g/day) between 28 and 63 days of age. Features of the marginal posterior distributions for ratios of genetic variance, variance owing to non-additive plus environmental permanent male effects and variance owing to common litter of birth effects with respect to phenotypic variance are reported. The correlations between sperm production traits and the selection criteria were also estimated. Three sets of two-trait analyses were performed, involving 12908 records of DG, 2329 ejaculates corresponding to 412 bucks and 14700 animals in pedigree file. The heritabilities (h²) of the semen traits were 0.13 ± 0.05, 0.08 ± 0.04 and 0.07 ± 0.03 for V, CN and PROD, respectively. The permanent environmental effects were lower than the corresponding values of h² and varied between 0.06 and 0.11. A favourable and moderate genetic correlation was observed between V and DG (0.36 ± 0.34; p > 0: 0.83), together with a non-favourable and moderate correlation between permanent environmental effects owing to common litter of birth for both traits (-0.35 ± 0.35; p < 0: 0.85). On the other hand, the correlation between male permanent environmental effects for semen traits and DG was moderate and non-favourable (-0.51 ± 0.29 with p < 0: 0.95 for DG-CN, and -0.31 ± 0.37 with p < 0: 0.79 for DG-PROD).

Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation

Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10-14 days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen-thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term. Further studies in subsequent embryo-foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects.

Efficiency of Repeated In Vivo Oocyte and Embryo Recovery After rhFSH Treatment in Rabbits

This study aims to assess the efficiency of in vivo oocyte and embryo recovery after a recombinant human FSH (rhFSH) treatment in rabbit does. Females were distributed in two experimental groups: donor does were treated with rhFSH (superovulation group) for 3 days prior to artificial insemination (embryo recovery) or ovulation induction (oocyte recovery) and does without treatment remained as the control group. Mature oocytes or embryos were collected with the laparoscopy technique 16 h after ovulation induction (oocytes) or 72 h after artificial insemination (embryos). Up to four recoveries were performed with each doe. Recovery efficiencies differed significantly between embryos (84%) and oocytes (58%). Yet, the recovery rates for the superovulation and control groups did not differ. The rhFSH group was associated with a significant increase (p < 0.05) in the number of oocytes and embryos recovered in comparison with the control group (10.2 +/- 1.0 and 14.3 +/- 1.2 vs 6.0 +/- 2.7 and 8.4 +/- 2.3 for oocytes and embryos, respectively). Results from this study indicate that repeated in vivo oocyte and embryo recovery from rhFSH superovulated does maximizes the number of oocytes or embryos collected from the same female.