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The application of ovum pickup-intracytoplasmic sperm injection (OPU-ICSI) is well established for the commercial equine in vitro production (IVP). There is a marked individual mare effect on the OPU-ICSI outcome, but little is known about the influence of the mare's health conditions. This study aimed to determine a potential association between the concentrations of interleukin-6 (IL-6) and reactive oxygen metabolites (d-ROMs) in the serum of the donor mare on the efficiency of equine IVP. Just before OPU, a blood sample was collected from 28 Warmblood donor mares, that were subjected to a routine OPU-ICSI program. The serum concentrations of IL-6 and d-ROMs were photometrically assayed using the equine Nori® (Genorise Scientific, USA) and Diacron® (Diacron International, Italy) kits, respectively. The kinetics of blastocyst development and the blastocyst rate (blastocysts/injected oocytes) were recorded. For the blastocyst forming mares, Pearson's and Spearman's correlation coefficients were calculated between the blastocyst rate and the serum concentration of IL-6 and d-ROMs. The serum concentrations were also compared between mares yielding at least one and mares yielding no blastocyst or between mares with fast (at day 7-8 post ICSI) and slow (≥ day 9 post ICSI)growing blastocysts using the independent t-test or Mann-Whitney U-test. The average blastocyst formation rate was 24.68±5.16 % and the average concentrations of IL-6 and d-ROMs were 519.59±157.08 pg/mL and 171.30±4.55 carratelli units (UCARR), respectively. Serum concentrations of IL-6 and d-ROMs, respectively, were not significantly correlated with blastocyst formation rates and not markedly different between the blastocyst forming (552.68±235.18 pg/mL and 168.36±5.56 UCARR) and non-forming (468.47±179.99 pg/mL and 175.85±7.89 UCARR) mares. Serum concentrations of d-ROMs were significantly lower in mares with fast growing (148.10±8.13 UCARR) compared to those with slow growing blastocysts (179.41±4.89 UCARR; P= 0.003). Taken together, the serum concentration of IL-6 and d-ROMs are not associated with the mares' ability to produce blastocysts after OPU-ICSI. Although it may be questioned whether a single sample is representative of the mare's health status, changes in serum metabolites related to oxidative stress at the time of oocyte retrieval may diminish the OPU-ICSI outcome via interfering with the embryonic development in mares. Funded by the Ministry of Higher Education of the Arab Republic of Egypt. [ABSTRACT FROM AUTHOR]

Improved efficiency of ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) resulted to a steep increase in the number of in vitro produced (IVP) equine embryos worldwide. These IVP blastocysts are routinely cryopreserved and both slow freezing and vitrification result in high pregnancy rates. The use of minimal volume cryodevices and fast, multistep warming protocols can be challenging for veterinary practitioners receiving vitrified embryos, but direct warming in embryo holding medium supports embryo viability (Canesin et al. Theriogenology. 2020;151:151-158). The aim of this study was to compare pregnancy rates in a clinical OPU-ICSI program using a three-step vitrification/warming protocol vs. a commercial vitrification kit and direct warming in embryo holding medium. Embryos were produced in autumn and winter of 2021-2022 by OPU-ICSI (Papas et al. Animals. 2021;11:2004). Day of vitrification (7-12 post ICSI) was similar for both vitrification groups. Blastocysts were vitrified in a minimal volume on a Cryolock (Irvine Scientific) following two methods: (1) a three-step protocol (n=21): holding in basal vitrification medium (BVM; DMEM-F12 with 20% FCS), vitrification in BVM with 1.5M ethylene glycol (5 min), followed by BVM with 7M ethylene glycol and 0.6M galactose (40 s), and warming in warming medium (WM; DPBS with 0.1% glucose,36 mg/L pyruvate, and 0.4% BSA) with 0.3M sucrose (1 min), followed by WM with 0.15M sucrose (5 min) and holding in WM (5 min), all performed at 38.2°C (Choi and Hinrichs. Theriogenology. 2017; 87:48-54); (2) direct warming (n=21): vitrification at 23°C with a commercial VIT-Kit (Minitube, Germany) in VS1 (5 min), VS2 (5 min) and VS3 (40 s), containing increasing concentrations of glycerol and/or ethylene glycol, based on Eldridge-Panuska et al. (Theriogenology. 2005; 63:1308-1319), and direct warming at 38°C for 5 min in 4 ml of Emcare holding medium (Spervital, The Netherlands). For both protocols, warmed embryos were washed four times in Emcare and transferred immediately to a day 4 recipient mare. The effect of vitrification method on pregnancy rate at 14 days and 25 days was determined using binomial logistic regression analyses. Pregnancy rates at 14 and 25 days were respectively 66.7% and 47.6% for the three-step method and 76.2% and 52.4% for the direct warming. No difference was found between the two methods (p=0.496 and p=0.882, respectively). In conclusion, vitrification and warming of IVP blastocysts using commercial media and direct warming supports pregnancy and provides an easy method for embryo warming and transfer by equine veterinary practitioners. [ABSTRACT FROM AUTHOR]

The number of equine embryos being produced increases yearly (37,094 +10% in 2021; newsletter IETS December 2022). The reasons include the growing popularity of in vitro embryo production (IVP), which increases the genetic yield from high quality mares, maximizes the use of expensive straws of semen from high quality stallions and enables IVP to be performed outside the reproductive season. On the 4th October 2022, a record price of €95,000 was paid for an embryo. However, despite the embryos' promising pedigrees, it remains unknown if transfer of such embryos will result in a healthy foal with the aspired phenotypical characteristics. This is largely because in horses, preimplantation genetic testing for aneuploidies and monogenic diseases or traits (PGT-A and PGT-M) remains underdeveloped especially when compared to humans. In addition, equine IVP has been reported to double early pregnancy losses, possibly because of an increased incidence of genetic errors. Here, we explored the possibility of concurrent genome-wide PGT-A and PGT-M in horses and assessed the incidence of chromosomal aberrations in IVP arrested or blastocyst stage embryos. We used GGP Equine SNP arrays (Illumina) with over 70,000 SNP positions to analyze whole-genome amplified DNA (Repli-G Single cell kit, Qiagen) from 14 trophectoderm biopsies and corresponding whole blastocysts and 26 individual cells from six embryos arrested at cleavage stage following IVP. Haplarithmisis (Zamani–Esteki et al. Am. J. Hum.Genet. 2015; 96:894-912) was applied to simultaneously determine the genome-wide copy number and haplotype. Five biopsies and seven single cells failed to give a result. From six arrested embryos, five had chromosomal errors in most of their cells, including (combinations of) triploidy and one or more monosomies and trisomies of meiotic and / or mitotic origin. Two biopsies showed a loss of maternal chromosome 28 and 31 respectively, which was confirmed in the corresponding blastocysts, suggesting meiotic errors. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found, suggesting a mitotic origin. Thus, aneuploidy and ploidy errors are highly prevalent in IVP arrested embryos compared to blastocysts. Like other mammals, these errors may contribute to embryonic arrest before or after clinical pregnancy detection. As a proof of principle, we determined the inherited 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) in one biopsy-embryo combination, confirming the potential of generic genome-wide PGT-M concurrent with PGT-A testing. Similar to humans, we postulate that embryo selection will improve embryo transfer success rates in horses and enable selection for traits and against diseases thus accelerating breeding programs. [ABSTRACT FROM AUTHOR]