Genomic tools are now available for most livestock species and are used routinely for genomic selection (GS) in cattle. One of the most important developments resulting from the introduction of genomic testing for dairy cattle is the application of reasonably priced low-density single nucleotide polymorphism technology in the selection of females. In this context, combining genome testing and reproductive biotechnologies in young heifers enables new strategies to generate replacement and elite females in a given period of time. Moreover, multiple markers have been detected in biopsies of preimplantation stage embryos, thus paving the way to develop new strategies based on preimplantation diagnosis and the genetic screening of embryos. Based on recent advances in GS, the present review focuses on new possibilities inherent in reproductive technologies used for commercial purposes and in genetic schemes, possible side effects and beneficial impacts on reproductive efficiency. A particular focus is on the different steps allowing embryo genotyping, including embryo micromanipulation, DNA production and quality assessment.
Recently, ProAKAP4 has been described as a pertinent indicator of sperm quality in humans, pigs, and stallions. In knockout mouse models lacking AKAP4 expression, the male mice were infertile. As high proAKAP4 levels were significantly correlated with a lower proportion of abortions in intrauterine insemination settings in human reproduction, proAKAP4 could be considered a pertinent new sperm parameter for assessing embryo quality. Our main goal was to assess the proAKAP4 concentrations in Holstein bull semen for comparison with the motility sperm parameters and fertility outcomes in post-thawed conditions. Straws issued from 52 ejaculates from 13 bulls, retrospectively identified with known nonreturn rates (NRR) as a fertility indicator, were provided by Evolution XY. Expression of ProAKAP4 and AKAP4 was assessed using enzyme-linked immunosorbent assay, western blotting, flow cytometry, and microscopy methods. Using the Bull 4MID kit (4BioDx), striking variations in proAKAP4 concentrations were observed independently of the classic sperm parameters that were measured using computer-assisted semen analysis. A mean proAKAP4 concentration of 44.42ng per 10 million spermatozoa was obtained through all our series. Interestingly, the variations in proAKAP4 concentrations were positively correlated with progressive motility and with the linearity coefficient parameter. Furthermore, the post-thawed concentrations of proAKAP4 were significantly higher in bulls with a higher NRR in a field study of more than 190 000 AI. We then demonstrated for the first time a correlation between the semen concentration of proAKAP4 and NRR (P=0.05) in bulls. Threshold values of proAKAP4 were then determined, with good values being between 25 and 60ngmL−1. Below 25ngmL−1, the sperm were of poor quality. The proportion of functional spermatozoa (i.e. spermatozoa expressing proAKAP4 in ejaculates) was assessed using flow cytometry. We observed that the cell debris and dead spermatozoa were never immunolabeled with proAKAP4 antibodies. On testis tissue sections, proAKAP4 was expressed only from the spermatids stages up to the ejaculated spermatozoa, being influenced by external factors and reflecting good spermatogenesis. Our preliminary study highlighted the pertinence of proAKAP4 in assessing sperm quality in bulls. It could be interesting to further analyse the effect of proAKAP4 level of expression on capacitation and IVF. As high levels of proAKAP4 were significantly correlated with fertility rates and with progressive motility, proAKAP4 could be proposed as a predictive marker of bull fertility and could be further investigated to evaluate the quality of invitro-produced embryos.
Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.
Bulls obtained by somatic cell nuclear transfer (SCNT) have proved to develop normally, produce sperm, and be fertile (Shiga et al. 2005 Theriogenology 64, 334–343; Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799). However, due to epigenetic variability encountered with clones, it cannot be excluded that major alterations may affect the X chromosome and especially the dosage of X inactivation in female embryos. This could result in a deviation of the sex ratio in offspring. To test this hypothesis, the sperm from a cloned bull was used in IVF to assess its ability to induce a different proportion of male and female embryos when compared to results obtained with the sperm of the original donor before cloning. In the present experiment, semen was collected twice weekly during 3 months from a 3-year-old cloned bull of the Charolais breed (H-2) and frozen-stored. Samples from 3 different ejaculates were used in comparison with control frozen straws from the original bull (H-0), prepared at the same age, to produce in vitro embryos. A total of 6 replicate IVF experiments were performed on the same batches of in vitro-matured oocytes (n = 654) using the standard swim-up and IVF technique in the laboratory. Twenty hours after insemination, presumptive zygotes were vortexed and cultured for 7 days in microdrops of B2 medium with Vero cells. Fertilization, cleavage, and blastocyst formation was assessed, and by Day 7, all of the blastocysts were individually frozen after grading. Two groups of 40 representative blastocysts derived from each bull (clone or donor) were used to determine the sex ratio using the sexing kit developed by UNCEIA R&D (Maisons-Alfort, France). Percentages between groups were compared by chi-square test. Semen from the cloned bull resulted in significantly lower fertilization and cleavage rates than that from the original donor (82.7% and 70% vs. 94.6% and 91%, respectively; P < 0.01), but further in vitro development was not different, as the proportions of blastocysts/cleaved were, respectively, 31.5% (74/235) vs. 38.7% (112/290). Sex determination was achieved in 79/80 of the in vitro-produced embryos and indicated that 55% of the blastocysts derived from the clone were male (22/40) and 45% female (18/40). This was not different from the proportion of male (61.5%, 24/39) and female (38.5%, 15/39) embryos in the group derived from semen of the original donor bull. In conclusion, these preliminary results indicate that the semen from this cloned Charolais bull has the same potential for in vitro embryo production as its original cell donor, and there is no evidence that SCNT could induce any deviation of the sex ratio. This observation needs to be confirmed with other sets of cloned bulls. (Shiga et al. 2005 Theriogenology 64, 334–343. Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799.)
The LH peak in cattle is the most precise event for predicting ovulation beginning 24 h later, and thus, AI time. Previous studies demonstrated that embryo production was improved when AI was conducted 12 h before ovulation; that is, 12 h after LH peak. This study aimed to evaluate the benefit of LH peak monitoring with Predi′Bov® (ReproPharm®, Nouzilly, France) following superstimulation in order to optimize numbers of viable embryos (VE). Predi′Bov® is a rapid (40 min) and easy to use on-farm test allowing LH peak detection from a few drops of blood. The test was also used to estimate the variability in the time of the LH peak and onset of oestrus. This study was conducted by the embryo transfer teams of 3 French cooperatives, in collaboration with UNCEIA. Forty heifers in stations (Creavia, Midatest) and 23 cows on farms (GEN′Iatest) were superstimulated by 8 injections IM of Stimufol® or Pluset® (FSH1 to FSH8) over 4 days in 2011–2012. Donor station heifers were treated twice in a Latin square design with a reference protocol where AI was conducted 12 and 24 h after onset of oestrus, and in an experimental protocol where AI was conducted 12 and 24 h after a positive Predi′Bov® test. Semen of different sires was used for both protocols. The Predi′Bov® test was carried out on 3 blood samples (BS1,2,3) collected every 6 h beginning at FSH7 in stations and FSH8 on farms to detect the earliest LH peaks. To determine late LH peaks, Predi′Bov® test was carried out on BS4 collected 24 h after FSH8. Univariate statistical analysis was performed to look at the relationship between qualitative (chi-square) and quantitative (t-test) variables. The difference was considered significant when P < 0.05. The Predi′Bov® test showed that 37.5% (15/40) of LH peaks occurred during the last day of FSH treatment (BS1 or BS2) in stations and 26.1% (6/23) at BS1 on farms. At Creavia station (n = 24), the LH peak was detected anytime from 24 h before to 9 h after the onset of oestrus. In stations, the VE percentage did not differ whether AI was done following oestrus or LH peak detection (63.1% and 61.8% in reference and experimental protocols, respectively). In stations, the VE percentage from 9 females with an LH peak detected at FSH7 (BS1; 41%) in the reference protocol did not differ from the experimental protocol (50%). On farms, the VE percentage was numerically higher but not significant in the experimental protocol (65.4%, n = 16) compared to the reference protocol (47.2%, n = 7). Further investigations are needed, taking into account the effect of collection rank, sire, and female effects, to confirm the trends shown by these results. In conclusion, Predi′Bov® can be used as easily on farms as in stations. Its use allows the detection of animals that have early or late LH peaks, which in turn provides the opportunity of carrying out AI at the optimal time for such females.
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