The rate of de novo structural variation is increased in in vitro-produced offspring and preferentially affects the paternal genome.

Assisted reproductive technologies (ARTs), including in vitro maturation and fertilization (IVF), are increasingly used in human and animal reproduction. Whether these technologies directly affect the rate of de novo mutation (DNM), and to what extent, has been a matter of debate. Here we take advantage of domestic cattle, characterized by complex pedigrees that are ideally suited to detect DNMs and by the systematic use of ART, to study the rate of de novo structural variation (dnSV) in this species and how it is impacted by IVF. By exploiting features of associated de novo point mutations (dnPMs) and dnSVs in clustered DNMs, we provide strong evidence that (1) IVF increases the rate of dnSV approximately fivefold, and (2) the corresponding mutations occur during the very early stages of embryonic development (one- and two-cell stage), yet primarily affect the paternal genome.

Screening of in vitro-produced cattle embryos to assess incidence and characteristics of unbalanced chromosomal aberrations.

In cattle, pregnancy rates of in vitro-produced embryos are lower than those of in vivo-produced embryos. One of the reasons may be the increase in chromosomal aberrations due to in vitro maturation and fertilization of the oocyte. Currently, embryo transfer is commonly applied in nucleus cattle breeding programs, and the embryos are genotyped for genomic selection. Therefore, intensity data from SNP arrays can be exploited for preimplantation genetic testing by screening the intensity data of the embryos for unbalanced chromosomal aberrations. A total of 558 stage 8 Dutch Holstein embryos genotyped with SNP arrays were screened in an observational study in retrospect. We found a 5% incidence rate of unbalanced chromosomal aberrations (aneuploidy and ploidy issues) among 430 successfully genotyped cattle embryos. The 22 affected embryos showed either aneuploidy or ploidy issues; monosomy was most frequently observed (14/22). In most cases (16/19) the maternal chromosome or chromosomes were lost or gained. One of the monosomy cases gave rise to a live-born fully diploid individual, suggesting mosaicism. Given that embryo genotypes are readily available, monitoring incidence can easily be applied. Moreover, selection for euploid embryos may improve pregnancy rates for in vitro embryo transfer.

Development of resilience indicator traits based on daily step count data for dairy cattle breeding.

BACKGROUND: Resilient animals are minimally affected by disturbances, such as diseases and heat stress, and quickly recover. Daily activity data can potentially indicate resilience, because resilient animals likely keep variations due to disturbances that threat animal homeostasis at a low magnitude. We used daily step count of cows to define resilience indicators based on theory, exploratory analysis and literature, and then investigated if they can be used to genetically improve resilience by estimating heritability and repeatability, and genetic associations with other resilience-related traits, i.e. health traits, longevity, fertility, and body condition score (BCS). RESULTS: Two groups of resilience indicators were defined: indicators describing (1) mean step count at different lactation stages for individual cows, and (2) fluctuations in step count from individual step count curves. Heritability estimates were highest for resilience indicators describing mean step count, from 0.22 for the 2-week period pre-partum to 0.45 for the whole lactation. High mean step count was consistently, but weakly, genetically correlated with good health, fertility, and longevity, and high BCS. Heritability estimates of resilience indicators describing fluctuations ranged from 0.01 for number of step count drops to 0.15 for the mean of negative residuals from individual curves. Genetic correlations with health traits, longevity, fertility, and BCS were mostly weak, but were moderate and favorable for autocorrelation of residuals (- 0.33 to - 0.44) and number of step count drops (- 0.44 to - 0.56) with hoof health, fertility, and BCS. Resilience indicators describing variability of residuals and mean of negative residuals showed strong genetic correlations with mean step count (0.86 to 0.95, absolute), which suggests that adjustment for step count level is needed. After adjustment, 'mean of negative residuals' was highly genetically correlated with hoof health, fertility, and BCS. CONCLUSIONS: Mean step count, autocorrelation and mean of negative residuals showed most potential as resilience indicators based on resilience theory, heritability, and genetic associations with health, fertility, and body condition score. Other resilience indicators were heritable, but had unfavorable genetic correlations with several health traits. This study is an important first step in the exploration of the use of activity data to breed more resilient livestock.

A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle.

Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a lead SNP, as a promising candidate variant. By implementing a fine-mapping and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.

Genetic architecture of individual variation in recombination rate on the X chromosome in cattle.

Meiotic recombination is an essential biological process that ensures proper chromosome segregation and creates genetic diversity. Individual variation in global recombination rates has been shown to be heritable in several species, and variants significantly associated with this trait have been identified. Recombination on the sex chromosome has often been ignored in these studies although this trait may be particularly interesting as it may correspond to a biological process distinct from that on autosomes. For instance, recombination in males is restricted to the pseudo-autosomal region (PAR). We herein used a large cattle pedigree with more than 100,000 genotyped animals to improve the genetic map of the X chromosome and to study the genetic architecture of individual variation in recombination rate on the sex chromosome (XRR). The length of the genetic map was 46.4 and 121.2 cM in males and females, respectively, but the recombination rate in the PAR was six times higher in males. The heritability of CO counts on the X chromosome was comparable to that of autosomes in males (0.011) but larger than that of autosomes in females (0.024). XRR was highly correlated (0.76) with global recombination rate (GRR) in females, suggesting that both traits might be governed by shared variants. In agreement, a set of eleven previously identified variants associated with GRR had correlated effects on female XRR (0.86). In males, XRR and GRR appeared to be distinct traits, although more accurate CO counts on the PAR would be valuable to confirm these results.

High temperature-humidity index compromises sperm quality and fertility of Holstein bulls in temperate climates.

Rising temperatures caused by climate change have adverse effects on cattle physiology, welfare, health, and reproduction. Heat stress in cows affects the oocyte and embryo directly through heat shock on cellular function. Fewer data are available on the effect of high temperatures on male fertility. Temperature-humidity index (THI) is a measure for assessing the risk of heat stress that combines the effects of temperature and humidity. The aim of this study was to determine the relationship between THI and fresh or frozen-thawed sperm quality of Holstein bulls kept in temperate climates. Bull sperm data of 29,170 ejaculates from 933 bulls collected at 3 Dutch artificial insemination centers between 2015 and 2018 were evaluated. The assessed variables included total sperm motility and morphology of fresh semen, and total sperm motility, morphology, and progressive motility of frozen semen 0 and 3 h after thawing. In addition, 56-d nonreturn rates were analyzed. The assessed effects were season and THI on the day of semen collection and during spermatogenesis (30 d before collection), bull, age of bull, year, and location. Bulls were divided into 2 categories according to their age: young (<36 mo) and older (>36 mo). Overall sperm quality of young bulls improved as age increased. No effect of THI on fresh sperm variables was observed in either young or older bulls. However, high THI at spermatogenesis negatively affected the cryotolerance of sperm cells. Sperm cells from young and older bulls showed a pronounced decrease (14-18%) of the assessed variables 3 h after thawing after the increase of THI during spermatogenesis in autumn. Remarkably, older bulls were more sensitive to THI at spermatogenesis compared with semen collection, showing up to a 3.8 times higher negative effect on frozen sperm quality. However, an elevated THI at semen collection produced a tendency toward decreased 56-d nonreturn rates as the age of the bull increased. Although this decrease was up to 4%, rising temperatures may still cause important economic losses in the future. For the first time, the present study confirmed that climate compromises not only sperm quality, but also dairy bull fertility.

Functional and population genetic features of copy number variations in two dairy cattle populations.

BACKGROUND: Copy Number Variations (CNVs) are gain or loss of DNA segments that are known to play a role in shaping a wide range of phenotypes. In this study, we used two dairy cattle populations, Holstein Friesian and Jersey, to discover CNVs using the Illumina BovineHD Genotyping BeadChip aligned to the ARS-UCD1.2 assembly. The discovered CNVs were investigated for their functional impact and their population genetics features. RESULTS: We discovered 14,272 autosomal CNVs, which were aggregated into 1755 CNV regions (CNVR) from 451 animals. These CNVRs together cover 2.8% of the bovine autosomes. The assessment of the functional impact of CNVRs showed that rare CNVRs (MAF < 0.01) are more likely to overlap with genes, than common CNVRs (MAF ≥ 0.05). The Population differentiation index (Fst) based on CNVRs revealed multiple highly diverged CNVRs between the two breeds. Some of these CNVRs overlapped with candidate genes such as MGAM and ADAMTS17 genes, which are related to starch digestion and body size, respectively. Lastly, linkage disequilibrium (LD) between CNVRs and BovineHD BeadChip SNPs was generally low, close to 0, although common deletions (MAF ≥ 0.05) showed slightly higher LD (r2 = ~ 0.1 at 10 kb distance) than the rest. Nevertheless, this LD is still lower than SNP-SNP LD (r2 = ~ 0.5 at 10 kb distance). CONCLUSIONS: Our analyses showed that CNVRs detected using BovineHD BeadChip arrays are likely to be functional. This finding indicates that CNVs can potentially disrupt the function of genes and thus might alter phenotypes. Also, the population differentiation index revealed two candidate genes, MGAM and ADAMTS17, which hint at adaptive evolution between the two populations. Lastly, low CNVR-SNP LD implies that genetic variation from CNVs might not be fully captured in routine animal genetic evaluation, which relies solely on SNP markers.

Exposing dairy bulls to high temperature-humidity index during spermatogenesis compromises subsequent embryo development in vitro.

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P < 0.001) was observed in the total blastocyst rates at day 7 post-insemination (15.3 vs 20.9%) and day 8 (23.2 vs 29.6%) in the high THI compared to the low THI group, respectively. There were no differences in the relative abundance of candidate transcripts examined. In conclusion, sperm samples from dairy bulls obtained during a period with higher THI had reduced viability and led to a decrease in blastocyst development and delayed hatching, compared to semen collected during a period with low THI.

In vitro production of bovine embryos derived from individual donors in the Corral® dish.

BACKGROUND: Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral® dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral® dish, each donor group in a separate quadrant. RESULTS: In two experiments, the Corral® dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral® dish used during IVM and IVC than when only used during IVM (12.9% ± 2.10 versus 22.8% ± 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination. CONCLUSIONS: In the present study, the Corral® dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral® dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral® dish is used during IVM and IVC.

Coding and noncoding variants in HFM1, MLH3, MSH4, MSH5, RNF212, and RNF212B affect recombination rate in cattle.

We herein study genetic recombination in three cattle populations from France, New Zealand, and the Netherlands. We identify 2,395,177 crossover (CO) events in 94,516 male gametes, and 579,996 CO events in 25,332 female gametes. The average number of COs was found to be larger in males (23.3) than in females (21.4). The heritability of global recombination rate (GRR) was estimated at 0.13 in males and 0.08 in females, with a genetic correlation of 0.66 indicating that shared variants are influencing GRR in both sexes. A genome-wide association study identified seven quantitative trait loci (QTL) for GRR. Fine-mapping following sequence-based imputation in 14,401 animals pinpointed likely causative coding (5) and noncoding (1) variants in genes known to be involved in meiotic recombination (HFM1, MSH4, RNF212, MLH3, MSH5) for 5/7 QTL, and noncoding variants (3) in RNF212B for 1/7 QTL. This suggests that this RNF212 paralog might also be involved in recombination. Most of the identified mutations had significant effects in both sexes, with three of them each accounting for ∼10% of the genetic variance in males.

The 80-kb DNA duplication on BTA1 is the only remaining candidate mutation for the polled phenotype of Friesian origin.

BACKGROUND: The absence of horns, called polled phenotype, is the favored trait in modern cattle husbandry. To date, polled cattle are obtained primarily by dehorning calves. Dehorning is a practice that raises animal welfare issues, which can be addressed by selecting for genetically hornless cattle. In the past 20 years, there have been many studies worldwide to identify unique genetic markers in complete association with the polled trait in cattle and recently, two different alleles at the POLLED locus, both resulting in the absence of horns, were reported: (1) the Celtic allele, which is responsible for the polled phenotype in most breeds and for which a single candidate mutation was detected and (2) the Friesian allele, which is responsible for the polled phenotype predominantly in the Holstein-Friesian breed and in a few other breeds, but for which five candidate mutations were identified in a 260-kb haplotype. Further studies based on genome-wide sequencing and high-density SNP (single nucleotide polymorphism) genotyping confirmed the existence of the Celtic and Friesian variants and narrowed down the causal Friesian haplotype to an interval of 145 kb. RESULTS: Almost 6000 animals were genetically tested for the polled trait and we detected a recombinant animal which enabled us to reduce the Friesian POLLED haplotype to a single causal mutation, namely a 80-kb duplication. Moreover, our results clearly disagree with the recently reported perfect co-segregation of the POLLED mutation and a SNP at position 1 390 292 bp on bovine chromosome 1 in the Holstein-Friesian population. CONCLUSION: We conclude that the 80-kb duplication, as the only remaining variant within the shortened Friesian haplotype, represents the most likely causal mutation for the polled phenotype of Friesian origin.

A deletion in the bovine FANCI gene compromises fertility by causing fetal death and brachyspina.

Fertility is one of the most important traits in dairy cattle, and has been steadily declining over the last decades. We herein use state-of-the-art genomic tools, including high-throughput SNP genotyping and next-generation sequencing, to identify a 3.3 Kb deletion in the FANCI gene causing the brachyspina syndrome (BS), a rare recessive genetic defect in Holstein dairy cattle. We determine that despite the very low incidence of BS (<1/100,000), carrier frequency is as high as 7.4% in the Holstein breed. We demonstrate that this apparent discrepancy is likely due to the fact that a large proportion of homozygous mutant calves die during pregnancy. We postulate that several other embryonic lethals may segregate in livestock and significantly compromise fertility, and propose a genotype-driven screening strategy to detect the corresponding deleterious mutations.