Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Oestrus is the period in the sexual cycle of female mammals where they become most receptive to mating and are most fertile. Efficient detection of oestrus is a key component in successful reproductive livestock management programmes. Oestrus detection in cattle is most often performed by visual observation, such as mounting behaviour and standing heat, to facilitate more successful prediction of optimal time points for artificial insemination. This time-consuming method requires a skilled, diligent observer. Biological measurements using easily accessible biomolecules in the cervico-vaginal mucus could provide an alternative strategy to physical methods of oestrus detection, providing an inexpensive means of rapidly and accurately assessing the onset of oestrus. In this study, glycosylation changes in cervico-vaginal mucus from three heifers following oestrus induction were investigated as a proof of concept to assess whether potential glycosylation-based trends could be useful for oestrus stage indication. Mucus collected at different time points following oestrus induction was immobilised in a microarray format and its glycosylation interrogated with a panel of fluorescently labelled lectins, carbohydrate-binding proteins with different specificities. Individual animal-specific glycosylation patterns were observed, however each pattern followed a similar trend around oestrus. This unique oestrus-associated glycosylation was identified by a combination of relative binding of the lectins SNA-I and WFA for each animal. This alteration in cervico-vaginal mucus glycosylation could potentially be exploited in future to more accurately identify optimal fertilisation intervention points compared to visual signs. More effective oestrus biomarkers will lead to more successful livestock reproductive programmes, decreasing costs and animal stress.

Characterization of Breed Specific Differences in Spermatozoal Transcriptomes of Sheep in Australia.

Reduced reproductive efficiency results in economic losses to the Australian sheep industry. Reproductive success, particularly after artificial insemination, is dependent on a number of contributing factors on both ewe and ram sides. Despite considerable emphasis placed on characterising ewe side contributions, little emphasis has been placed on characterising ram side contributions to conception success. Over 14,000 transcripts are in spermatozoa of other species, which are transferred to the ova on fertilisation. These transcripts conceivably influence early embryonic development and whether conception is successful. Semen was collected (n = 45) across three breeds; Merino, Dohne, and Poll Dorset. Following collection, each ejaculate was split in two; an aliquot was assessed utilising Computer Assisted Semen Analysis (CASA) and the remaining was utilised for RNA extraction and subsequent next-generation sequencing. Overall, 754 differentially expressed genes were identified in breed contrasts and contrast between ejaculates of different quality. Downstream analysis indicated that these genes could play significant roles in a broad range of physiological functions, including maintenance of spermatogenesis, fertilisation, conception, embryonic development, and offspring production performance. Overall results provide evidence that the spermatozoal transcriptome could be a crucial contributing factor in improving reproductive performance as well as in the overall productivity and profitability of sheep industries.

Genotyping strategies of selection candidates in livestock breeding programmes.

Benefits of genomic selection (GS) in livestock breeding operations are well known particularly where traits are sex-limited, hard to measure, have a low heritability and/or measured later in life. Sheep and beef breeders have a higher cost:benefit ratio for GS compared to dairy. Therefore, strategies for genotyping selection candidates should be explored to maximize the economic benefit of GS. The aim of the paper was to investigate, via simulation, the additional genetic gain achieved by selecting proportions of male selection candidates to be genotyped via truncation selection. A two-trait selection index was used that contained an easy and early-in-life measurement (such as post-weaning weight) as well as a hard-to-measure trait (such as intra-muscular fat). We also evaluated the optimal proportion of female selection candidates to be genotyped in breeding programmes using natural mating and/or artificial insemination (NatAI), multiple ovulation and embryo transfer (MOET) or juvenile in vitro fertilization and embryo transfer (JIVET). The final aim of the project was to investigate the total dollars spent to increase the genetic merit by one genetic standard deviation (SD) using GS and/or reproductive technologies. For NatAI and MOET breeding programmes, females were selected to have progeny by 2 years of age, while 1-month-old females were required for JIVET. Genomic testing the top 20% of male selection candidates achieved 80% of the maximum benefit from GS when selection of male candidates prior to genomic testing had an accuracy of 0.36, while 54% needed to be tested to get the same benefit when the prior selection accuracy was 0.11. To achieve 80% of the maximum benefit in female, selection required 66%, 47% and 56% of female selection candidates to be genotyped in NatAI, MOET and JIVET breeding programmes, respectively. While JIVET and MOET breeding programmes achieved the highest annual genetic gain, genotyping male selection candidates provides the most economical way to increase rates of genetic gain facilitated by genomic testing.

Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model.

PURPOSE: Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes. METHODS: Semen was collected from 40 bucks, incubated at 38 °C for 24 h, and the rate of sperm DNA fragmentation (rSDF) was determined using the sperm chromatin dispersion assay. Males were allocated into high rSDF (>0.5 units of increase per hour) or low rSDF (<0.5 units of increase per hour) groups. High and low rSDF semen samples were sequentially artificially inseminated into the same doe to reduce female factor variability, and pregnancy outcomes were recorded. RESULTS: While there was no difference in SDFs between rSDF groups immediately after collection (T0), differences were significant after 2 h of incubation; SDFs determined at collection and rSDF behaved as independent characters (Pearson correlation = 0.099; P = 0.542). Following artificial insemination, the rate of stillborn pups was significantly higher in does inseminated by males with a high rSDF (14/21) compared to those with low rSDF (15/6); (contingency χ(2) 5.19; p = 0.022). The risk of stillborn when low rSDF rabbits were used for insemination was 0.16, but increased to 0.36 when high rSDF animals were used (odds ratio = 2.85; 95 % confidence interval = 1.4-2.7). CONCLUSION(S): Dynamic assessment of SDF coupled with natural multiple ovulation, high fecundity of the rabbit and control over female factor influence, provided a useful experimental model to demonstrate the adverse effect of reduced sperm DNA longevity on reproductive outcome.

Genetic parameters of rabbit semen traits and male fertilising ability.

This study aimed to estimate genetic parameters for rabbit semen production, semen characteristics and fertilising ability following artificial insemination. It involved five successive batches of 30-36 bucks each, 22 weeks of semen collection, and 11 weeks of semen recording per batch. Semen analyses were based on 2312 ejaculates. A total of 2019 inseminations were performed on 674 females with semen from 236 ejaculates from 128 bucks. Heritability estimates of semen traits ranged from 0.05 to 0.18. At approximately 0.05-0.06 for pH, volume and mass motility, they were higher for concentration (0.10) and the total number of sperms per ejaculate (0.12), and even higher for motility traits based on computer-assisted semen analysis. The percentage of motile sperms had the highest heritability (0.18) and appeared to be a good candidate criterion to select for both sperm number and motility. The heritability estimates were close to zero for all three criteria of fertilising ability: fertility (F), prolificacy (live births, LB) and their product (LB per insemination). A permanent environmental effect of the male seemed to be higher for LB (0.04) than for F (0.01). The rabbit does accounted for approximately 10% of the variance of the three criteria. With respect to the female, the male contribution was negligible for fertility and in a ratio of 4-10 for the number of live births. In our experimental conditions, prolificacy would thus be more highly influenced by the buck than fertility.

Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts.

Efficient Boar Semen Production and Genetic Contribution: The Impact of Low-Dose Artificial Insemination on Fertility.

Diluting semen from high fertile breeding boars, and by that inseminating many sows, is the core business for artificial insemination (AI) companies worldwide. Knowledge about fertility results is the reason by which an AI company can lower the concentration of a dose. Efficient use of AI boars with high genetic merit by decreasing the number of sperm cells per insemination dose is important to maximize dissemination of the genetic progress made in the breeding nucleus. However, a potential decrease in fertility performance in the field should be weighed against the added value of improved genetics and, in general, is not tolerated in commercial production. This overview provides some important aspects that influence the impact of low-dose AI on fertility: (i) the importance of monitoring field fertility, (ii) the need for accurate and precise semen assessment, (iii) the parameters that are taken into account, (iv) the application of information from genetic and genomic selection and (v) the optimization when using different AI techniques. Efficient semen production, processing and insemination in combination with increasing use of genetic and genomic applications result in maximum impact of genetic trend.

¿Es ético congelar ovocitos para retrasar la maternidad por motivos laborales? / Is it ethical to freeze oocytes to delay childbearing for employment?

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Niveles de PAPP-A y fßhCG en gestantes gemelares bicoriónicas concebidas por técnicas de reproducción asistida (FIV-IAH) y sus implicaciones en el cálculo del riesgo del cribado prenatal del primer trimestre / PAPP-A and fBhCG levels in dichorionic twin pregnancies conceveid by assisted reproductive technologies (IVF-AIH) and their implications to calculate the prenatal risk on first trimestry

Objetivo: Evaluar los niveles de proteína plasmática placentaria (PAPP-A) y fracción libre de la βhCG (fßhCG) en gestaciones gemelares bicoriónicas concebidas mediante técnicas de reproducción asistida tipo fertilización in vitro (FIV) o inseminación artificial (IA) en nuestra población. Posterior cálculo de los factores de corrección necesarios a aplicar para el screening de primer trimestre en gestantes gemelares concebidas mediante FIV. Población en estudio: Se han estudiado 171 gestantes gemelares. Un grupo compuesto por 71 gestaciones bicoriónicas concebidas mediante FIV, con una edad media de 36,3 años; un grupo control de 76 gestaciones bicoriónicas concebidas por gestación espontánea, con una edad media de 31,8 años; y un último grupo de 24 gestaciones bicoriónicas concebidas por IA, con una edad media de 32,9 años. Resultados: Las gestantes gemelares bicoriónicas concebidas mediante FIV respecto el grupo control, concebidas espontáneamente, presentan unos niveles estadísticamente inferiores de PAPP-A (2,17 MoM versus 2,41 MoM, p<0,008) y valores superiores no significativos de fßhCG (2,33 MoM versus 2,05 MoM, p=0,144). Las medias de la translucencia nucal (Tn) entre ambos grupos tampoco muestran diferencias significativas (p=0,178). Esta misma tendencia se observa en los valores de PAPPA y fßhCG para las gestaciones concebidas mediante IA, pero con un número menor de gestantes no significativo. Conclusiones: Las mujeres con gestaciones gemelares concebidas por técnicas de reproducción asistida han sido sometidas a tratamientos para la estimulación de la ovulación. Este hecho se ha postulado como una de las causas que podrían explicar la presencia de valores de PAPP-A significativamente inferiores y de fßhCG superiores, aunque no significativos, a las gestantes gemelares espontáneas. Por ello se aconseja la elaboración de curvas propias con gestantes gemelares FIV, o en su defecto, el empleo de factores de corrección para disminuir este efecto. Se comprueba que con la aplicación de estos factores de corrección la tasa de falsos positivos de los gemelares bicoriónicos concebidos por FIV baja de 4,22% a 1,41% en nuestra población, con la consecuente disminución de biopsias de corion que son especialmente complejas en gestaciones gemelares (AU)

Objective: Evaluation of levels of PAPP-A and fßhCG in dichorionic twin pregnancies conceived by Assisted Reproductive Technology, i.e. In Vitro Fertilization (IVF) and Artificicial Insemination (AI) in our population. Calculate the necessary correction factors for first trimester screening in pregnancies conceived by IVF. Study population: One hundred seventyone twin pregnancies were considered within this study. One group was composed by 71 dichorionic twin pregnancies conceived by IVF with an average maternal age of 36.6 years. The control group was composed by 76 dichorionic twin pregnancies conceived spontaneously with an average maternal age of 31.8. A third group was composed by 24 dichorionic twin pregnancies conceived by AI with an average maternal age of 32.9 years. Results: Dichorionic twin pregnancies conceived by IVF present statistically significant lower values for PAPP-A than the control group (2.17 MoM versus 2.41 MoM, p<0.008) and higher values of fßhCG though this difference is not significant (2.33 MoM versus 2.05 MoM, p=0.144). In the other hand, average nuchal translucency (NT) does not show statistical differences between these two groups (p=0.178). Twin pregnancies conceived by AI present these same results tendencies for PAPP-A, fßhCG, and NT, with a lower number of patients not significant. Conclusions: Women with twin pregnancies conceived by Assisted Reproductive Technology (ART) have undergone treatments to induce ovulation. These treatments are proposed as one of the causes that may justify lower PAPP-A values and higher (though not significant) fßhCG values, when twin pregnancies conceived by ART are compared to spontaneously conceived twin pregnancies. Construction of normality curves for twin pregnancies conceived by IVF or calculation of correction factors is advised, in order to decrease this effect in the studied biochemical parameters. Applying these correction factors to our population data, the rate of false positives in first trimester screenings in dichorionic twin pregnancies conceived by IVF is decreased from a 4.22% to a 1.41%; this consequently implies a reduction of chorion biopsies, which are specially complicated in twin pregnancies (AU)

(Co)variance components, genetic parameters and annual trends for calf weights in a Brahman herd kept on floodable savanna

(Co)variance components and genetic parameters were estimated for body weights of an elite Brahman herd under a designed, supervised management and genetic program, including strategic artificial insemination (AI). Restricted maximum likelihood methods were used with a univariate animal model for birth weight (BW) and a bivariate model for weaning weight (205-day weight, 205W) and 18-month weight (548-day weight, 548W). Models included random animal direct and maternal genetic effects, maternal permanent environmental effect (c2), and sex-year-month of birth-age of dam and genetic group (identified and unidentified paternity), as fixed effects. Analysis A1 included all calves and analysis A2 included only those with identified sires. Of the 8,066 calves born, 36 were progeny of AI, 11 from single sire and 53 from multi-sire herds. They were born from 1985 to 1998, from 2559 dams and 146 sires (78 identified). Estimates of direct, maternal and total heritabilities from A1 for BW, 205W and 548W were: 0.23, 0.07 and 0.30; 0.08, 0.14 and 0.16; 0.16, 0.04 and 0.28, respectively. Corresponding estimates of direct maternal genetic correlations were 0.22, 0.07 and 0.86, and c2 estimates were 0.04, 0.14 and 0.04, respectively. Estimates of direct and maternal genetic, and permanent environmental correlations between 205W and 548W were: 0.66, 0.70 and 1.00. Variances and genetic parameters from A1 and A2 were, in general, very similar. Estimates of phenotypic, and direct and maternal genetic trends per year from A1 were: 0.393, 0.004 and 0.003 kg (BW), 3.367, 0.142 and 0.115 kg (205W), 1.813, 0.263 and 0.095 kg (548W). Estimates of direct and maternal genetic trends from A2 were: 0.033 and -0.002 kg (BW); 0.186 and 0.276 kg (205W); 0.471 and 0.136 kg (548W). The modern selection methods that have been used recently should be continued, with emphasis on the improvement of cow efficiency for sustainable beef production on floodable savanna combined with improved pasture

Should gamete donors be tested for spinal muscular atrophy?

OBJECTIVE: To report two cases of spinal muscular atrophy (SMA) after artificial insemination and to discuss why genetic screening of the disease may be justified in gamete donors. DESIGN: Case report. SETTING: Academic departments of genetics and obstetrics. PATIENT(S): A 32-year-old woman with two successive assisted pregnancies. INTERVENTION(S): Molecular studies of the SMN1 (survival motor neuron), the determining gene of the disease. MAIN OUTCOME MEASURE(S); Prenatal testing to detect a homozygous deletion of the SMN1 gene; carrier diagnosis by quantitative analysis to detect a single or double dose of exon 7 in the SMN1 gene. RESULT(S): After a first assisted pregnancy, an SMA child with a homozygous deletion of the SMN1 gene was born. In the second assisted pregnancy, using sperm from a different donor, a fetus with a homozygous deletion of SMN1 was detected. Carrier status in the donor was confirmed by a single dose of SMN1 in the quantitative analysis. CONCLUSION(S): Genetic screening of SMA carrier status by quantitative analysis of the SMN1 gene should be performed in gamete donors when the recipient is a known carrier. Cost-benefit analysis should be made to consider the inclusion of the test in prospective gamete donor programs.

(Co)variance components, genetic parameters and annual trends for calf weights in a Brahman herd kept on floodable savanna.

(Co)variance components and genetic parameters were estimated for body weights of an elite Brahman herd under a designed, supervised management and genetic program, including strategic artificial insemination (AI). Restricted maximum likelihood methods were used with a univariate animal model for birth weight (BW) and a bivariate model for weaning weight (205-day weight, 205W) and 18-month weight (548-day weight, 548W). Models included random animal direct and maternal genetic effects, maternal permanent environmental effect (c2), and sex-year-month of birth-age of dam and genetic group (identified and unidentified paternity), as fixed effects. Analysis A1 included all calves and analysis A2 included only those with identified sires. Of the 8,066 calves born, 36% were progeny of AI, 11% from single sire and 53% from multi-sire herds. They were born from 1985 to 1998, from 2559 dams and 146 sires (78 identified). Estimates of direct, maternal and total heritabilities from A1 for BW, 205W and 548W were: 0.23, 0.07 and 0.30; 0.08, 0.14 and 0.16; 0.16, 0.04 and 0.28, respectively. Corresponding estimates of direct maternal genetic correlations were 0.22, 0.07 and 0.86, and c2 estimates were 0.04, 0.14 and 0.04, respectively. Estimates of direct and maternal genetic, and permanent environmental correlations between 205W and 548W were: 0.66, 0.70 and 1.00. Variances and genetic parameters from A1 and A2 were, in general, very similar. Estimates of phenotypic, and direct and maternal genetic trends per year from A1 were: 0.393, 0.004 and 0.003 kg (BW), 3.367, 0.142 and 0.115 kg (205W), 1.813, 0.263 and 0.095 kg (548W). Estimates of direct and maternal genetic trends from A2 were: 0.033 and -0.002 kg (BW); 0.186 and 0.276 kg (205W); 0.471 and 0.136 kg (548W). The modern selection methods that have been used recently should be continued, with emphasis on the improvement of cow efficiency for sustainable beef production on floodable savanna combined with improved pasture.

Failure of founder transgenic male mice to transmit an attenuated HSV thymidine kinase transgene results from mosaicism and sperm competition.

Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.

Nuevos conseptos en espermiologia / New concepts in spermatology

Se establecen paràmetros, tècnicas y fòrmulas para una valoraciòn exacta del estudio animal y concomitantemente la forma de valorar de fertilidad masculina. Dicha mètadologia es la usada por el autor laboratorio del I.G.H.