Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection.

In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

A genome-wide association study of mare fertility in the Pura Raza Español horse.

Despite the economic importance of fertility for the horse industry, few efforts have been made to achieve a better understanding of the genetic mechanisms underlying its control. This is probably due to the difficulty of obtaining reliable phenotypes and the complexity of modelling the environmental and management factors. This work is novel in that we propose to use reproductive efficiency (RE) as an indicator of mare fertility. To achieve this, we performed a genome-wide association study in the Pura Raza Español horse aimed at identifying genomic variants, regions, and candidate genes associated with fertility in mares. The dataset included 819 animals genotyped with the Affymetrix Axiom™ Equine 670 K single-nucleotide polymorphisms (SNPs) Genotyping Array and the deregressed breeding values for RE trait, obtained using a ssBLUP model, employed as pseudo-phenotypic data. Our results showed 28 SNPs potentially associated with RE, which explained 87.19% of the genetic variance and 6.61% of the phenotypic variance. Those results were further validated in BayesB, showing a correlation between observed and predicted RE of 0.57. In addition, 15 candidate genes (HTRA3, SPIRE1, APOE, ERCC1, FOXA3, NECTIN-2, KLC3, RSPH6A, PDPK1, MEIOB, PAQR4, NM3, PKD1, PRSS21, IFT140) previously related to fertility in mammals were associated with the markers and genomic regions significantly associated with RE. To our knowledge, this is the first genome-wide association study performed on mare fertility.

Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data.

Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrated that the procedure is not affected by the SNP density of the array employed or by the inbreeding level of the individuals. Finally, the methodology proposed in this study could be performed in an open bioinformatic environment, thus permitting its integration as a flexible screening tool in diagnostic laboratories and genomic breeding programs.

Phenotypic and genetic analysis of reproductive traits in horse populations with different breeding purposes.

Reproductive traits have a major influence on the economic effectiveness of horse breeding. However, there is little information available. We evaluated the use of reproductive traits as selection criteria in official breeding programs to increase the reproductive efficiency of breeding studs, analysing 696 690 records from the pedigree data of eight Spanish horse populations, with different breeding purposes. The reproductive parameters studied in both sexes were age at first foaling (AFF), age at last foaling, average reproductive life and generational interval. In the females, the average interval between foaling (AIF) and interval between first and second foaling were also studied. There were clear differences between sexes and breeds, which may be due to management practices, breeding purposes and the status of the populations, rather than to differences in actual physiological conditions. Riding mares were the most precocious (AFF, 1937.64 to 2255.69 days) and had a more intensive reproductive use (AIF, 625.83 to 760.07 days), whereas sires used for meat production were the most precocious males (AFF, 1789.93 to 1999.75 days), although they had a shorter reproductive life (1564.34 to 1797.32 days). Heritabilities (0.02 to 0.42 in females and 0.04 to 0.28 in males) evidenced the genetic component of the reproductive traits, with Sport Horses having the higher average values. These results support the selection by AFF to improve reproductive aspects because of its medium-high heritability and its positive correlations with other important reproductive traits. The inclusion of the AIF is also recommended in sport populations, because this determines the length of the breaks between foaling and conditions the reproductive performance of the dams, as well as their selective intensity, genetic gain and genetic improvement. It is therefore an important economic parameter in breeding studs.

Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.

Prevalence of twin foaling and blood chimaerism in purebred Spanish horses.

Twin foaling is associated with chimaerism in several domestic species and is recognised in horses. In this study, 21,097 purebred Spanish (Pura Raza Español) horse births from the 2015 to 2016 breeding season were investigated for chimaerism. Twin foaled and chimaeric individuals were assessed on the basis of foaling records, short-tandem repeat (STR) parentage test results and a sex-linked STR-based technique. Fourteen twin pregnancies with 23 twin foals born alive were identified (0.066% twin foaling prevalence), including five blood chimaeric cases (21.7%; overall prevalence 0.011%), suggesting that this genetic condition is extremely low in horses. Furthermore, no true chimaeras were detected. This is the first large scale study analysing the occurrence of chimaerism in a horse population and the first assessment of twin foaling in purebred Spanish horses.

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN2) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN2 vapour; and P3 (rapid freezing) semen frozen immediately in LN2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN2 vapour.

Bovine thyroglobulin gene polymorphisms and their association with sexual precocity in Guzerat bulls.

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well-described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse.

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)-based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAX-linked (LEX026, LEX003, TKY38, TKY270 and UCDEQ502) and two ECAY-linked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner's syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders.

First case of sterility associated with sex chromosomal abnormalities in a jenny.

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.

DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy.

The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques.

This study compared the efficacy of simple sperm washing (SW), single-layer centrifugation (SLC) and modified swim-up (SU) techniques in the preparation of dog spermatozoa for cooling. Eighteen ejaculates, collected from three dogs (six per dog), were pooled (three ejaculates per pool) and divided into three aliquots: (1) one aliquot was washed and cooled at 5°C for 72h, considered as control (SW-control), (2) the second aliquot was selected by SLC through Androcoll-C and subsequently cooled in the same way as the SW-control samples (SLC-AC) and (3) the last aliquot was selected by a modified SU method with Androcoll-C and cooled as mentioned above (SU-AC). Assessment of sperm motility, sperm morphology, sperm membrane integrity and acrosome integrity were performed on aliquots of fresh semen and chilled-rewarmed samples. Sperm membrane integrity and progressive motility were significantly (PPP>0.05). The recovery rates were not significantly (P>0.05) different between SW-control, SLC-AC and SU-AC samples. Our results confirm that SU-AC may be a successful method for the preparation of dog spermatozoa for cooling.

Cryopreservation of canine semen after cold storage in a Neopor box: effect of extender, centrifugation and storage time.

The aim of this work was to assess the combined effect of sperm centrifugation, semen extender and storage time before freezing on post-thaw sperm quality and freezability on chilled stored canine semen in a Neopor box. Sperm parameters evaluated were total and progressive sperm motility by Computer-Assisted Sperm Analysis (CASA) and sperm viability and acrosome integrity using a triple fluorescent stain. Sperm quality and freezability indexes were also studied. First, the effect of centrifugation and two commercial extenders from Minitübe (Biladyl A and CaniPRO Freeze A) was evaluated in chilled semen after 24 and 45 hours of cold storage. No significant differences were observed between treatments in almost all the sperm parameters assessed. Secondly, chilled semen was frozen after 24 and 45 hours of cold storage in a Neopor box. The best results were obtained when semen was centrifuged, chilled with CaniPRO Freeze A and then frozen after 24 hours of cold storage, showing no differences in both post-thaw sperm quality and freezability in comparison with semen immediately frozen after collection. In conclusion, dog semen centrifuged after collection and extended with CaniPRO Freeze can be frozen after 24 hours of cold storage in a Neopor box, obtaining similar results to semen immediately frozen after collection.

The use of a novel combination of diagnostic molecular and cytogenetic approaches in horses with sexual karyotype abnormalities: a rare case with an abnormal cellular chimerism.

Sex chromosome aberrations are known to cause congenital abnormalities and unexplained infertility in horses. Most of these anomalies remain undiagnosed because of the complexity of the horse karyotype and the lack of specialized laboratories that can perform such diagnoses. On the other hand, the utilization of microsatellite markers is a technique widely spread in horse breeding, mostly because of their usage in parentage tests. We studied the usage of a novel combination of diagnostic approaches in the evaluation of a very uncommon case of chromosomal abnormalities in a Spanish purebred colt, primarily detected using a commercial panel of short tandem repeat (STR) makers. Based on these results, we performed a full cytogenetic analysis using conventional and fluorescent in situ hybridization techniques with individual Equus caballus chromosome X and Equus caballus chromosome Y painting probes. We also tested the presence of two genes associated with the sexual development in horses and an extra novel panel of eight microsatellite markers specifically located in the sex chromosome pair. This is the first case report of a leukocyte chimerism between chromosomally normal (64,XY) and abnormal (63,X0) cell lines in horses. Our results indicate that the use of the short tandem repeat markers as a screening technique and as a confirmation utilizing cytogenetic techniques can be used as a very interesting, easy, and nonexpensive diagnostic approach to detect chromosomal abnormalities in the domestic horse.

Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test.

The aims of this study were: 1) to assess the effect of freezing and thawing on dog sperm DNA fragmentation index (sDFI) using the sperm chromatin dispersion test (SCDt); and 2) to determine whether or not the sperm selection by single layer centrifugation (SLC) using Androcoll-C improves sperm DNA longevity in SLC-selected frozen-thawed dog semen samples. Semen samples were collected from 4 dogs using digital manipulation. After collection, ejaculates were pooled and cryopreserved following a standard protocol. Sperm motility and morphology were assessed before freezing and after thawing as a control for the cryopreservation method used. In experiment 1, sDFI was analyzed immediately before freezing and after thawing (baseline values), showing no significant differences between fresh and frozen-thawed semen samples. In experiment 2, frozen-thawed semen samples were processed or not by SLC using Androcoll-C and longevity of DNA were assessed in terms of sDFI after 24h of in vitro incubation at physiological temperature (38°C). The results showed low values of sDFI in SLC-selected semen in comparison to unselected samples. In conclusion, no effect of cryopreservation was observed on baseline values of dog sperm DNA fragmentation. Additionally, SLC-selection using Androcoll-C improved longevity of frozen-thawed sperm DNA assessed by the SCDt.

The use of molecular and cytogenetic methods as a valuable tool in the detection of chromosomal abnormalities in horses: a case of sex chromosome chimerism in a Spanish purebred colt.

Chromosomal abnormalities associated to sex chromosomes are reported as a problem more common than believed to be in horses. Most of them remain undiagnosed due to the complexity of the horse karyotype and the lack of interest of breeders and veterinarians in this type of diagnosis. Approximately 10 years ago, the Spanish Purebred Breeders Association implemented a DNA paternity test to evaluate the pedigree of every newborn foal. All candidates who showed abnormal or uncertain results are routinely submitted to cytogenetical analysis to evaluate the presence of chromosomal abnormalities. We studied the case of a foal showing 3 and even 4 different alleles in several loci in the short tandem repeat (STR) -based DNA parentage test. To confirm these results, a filiation test was repeated using follicular hair DNA showing normal results. A complete set of conventional and molecular cytogenetic analysis was performed to determine their chromosomal complements. C-banding and FISH had shown that the foal presents a sex chimerism 64,XX/64,XY with a cellular percentage of approximately 70/30, diagnosed in blood samples. The use of a diagnostic approach combining routine parentage QF-PCR-based STR screening tested with classical or molecular cytogenetic analysis could be a powerful tool that allows early detection of foals that will have a poor or even no reproductive performance due to chromosomal abnormalities, saving time, efforts and breeders' resources.