Research was carried out under field conditions on six foals of the Polish Konik breed born at the stud farm owned by the Research Station of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Popielno, Poland. The births took place at night, without complications for any offspring or dams and without any assistance or care from humans. All foals were born clinically healthy, and their dams did not show any signs of disease during pregnancy or after birth.
To estimate the structure and changes in the eye microbiome of newborn foals, a clinical investigation was conducted. The procedures performed in the project did not need the approval of the Institutional Animal Care and Use Committee, which was confirmed by the Local Ethical Committee at the University of Warmia and Mazury in Olsztyn with edict LKE.31.01.2020. Samples were taken longitudinally, not later than 12 h after delivery (time point I) and again after the first (time point II) and second (time point III) months of life (Supplementary Table 1). Samples from the left and right eye were individually collected from the ventral conjunctival fornix using sterile cotton swabs soaked in sterile 0.9% saline (Sarstedt, Copan, Brescia, Italy) (34 (link)). Special care was taken not to contaminate the swabs by contact with the eyelids or eyelashes. The left and right eye of each foal was swabbed once and the procedure was duplicated. A total of 24 swabs (4 per animal) were collected during each sampling. Specimens were quickly placed in cryogenic tubes at a temperature of −20°C for freezing and storage. After the conjunctival swab collection was completed, samples were shipped in an expanded polystyrene box with a cooler (at a temperature of 2–4°C) by courier directly for analysis (Genomed, Warsaw, Poland). The swabs were not frozen again. The same method of swabbing and storage is commonly used in human medicine to avoid contamination with other microbiota (3 (link)).
Genomic DNA was isolated immediately after delivery of samples using a Genomic Mini AX Bacteria kit (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s instructions, with an additional mechanical lysis of each sample effected by zircon balls in a FastPrep homogeniser (MP Biomedicals, Irvine, CA, USA), using a previously reported procedure (28 (link)). Concentration of DNA was measured through the fluorometric method, using a Qubit 4 fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA). The presence of bacterial DNA was confirmed in a qPCR reaction, using 1055F (5′-ATGGCTGTCGTCAGCT-3′) and 1392R (5′-ACGGGCGGTGTGTAC-3′) universal primers for 16S rRNA (5 (link)) and demineralised water as a negative control. The metagenomic analysis of bacteria and Archaea was based on the amplification of the V3–V4 hypervariable region (encompassing approximately 469 base pairs) of the 16S rRNA gene. For the amplification of the selected region and the preparation of DNA libraries, a pair of 341F and 785R primers and NEBNext Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) were used. For the measurement of DNA concentration, 1μL of the reaction mixture was taken immediately after the PCR. The unpurified product contained the reaction mixture with primers and primer dimers; therefore, the result for the negative control was not zero. Concentrations up to a value of approximately 1.5 ng/μL were considered acceptable. When the negative control was above this value (indicative of contamination with PCR reagents) the reaction was repeated. Arbitrarily, it was assumed that the lowest DNA concentration for the testing sample should be twice the minimal concentration of the negative control. Finally, purification of samples was performed with AMPure XP (Beckman Coulter, Indianapolis, IN, USA), followed by final DNA measurement by the fluorometric method on a Tecan reader (Männedorf, Switzerland). A PCR was carried out to index DNA in 50 μL reaction volumes. Next-generation sequencing (NGS) was performed on a MiSeq sequencer (Illumina, San Diego, CA, USA) in paired-end technology (PE300) by Genomed (Warsaw, Poland). MiSeq Reporter v.2.6 software (Illumina) was used for data analysis. To ensure the classification of reads at the species level, bioinformatic analysis was carried out with QIIME software (1 (link)), a semiquantitative approach to microbial ecology based on the SILVA v.138 database of reference sequences (24 (link)). Data analysis was performed of all sequences obtained from NGS and filtered. The diversity of the foals’ microbiota was analysed using alpha (Shannon and Simpson) and beta (Bray–Curtis) diversity indices (9 (link)). The diversity of microbial communities was compared in two ways: in all individuals between samplings and as a mean for the group between samplings, using sequence reads. All diversity indices were calculated for all six foals. Paired one-sided t-tests were used to verify statistically significant changes in biodiversity between collected samples. The calculations were made separately at the family, genus and species taxonomic levels. All calculations were performed in the MATLAB R2020a environment (MathLabs, Natick, MA, USA). Taxa present in amounts equal to or greater than 1% of the total identified DNA sequences at least in one of the individuals were classified as abundant, as proposed by Kim et al. (11 (link)). If the proportion of identified sequences was <1%, taxa were classified as nonabundant.
To estimate the structure and changes in the eye microbiome of newborn foals, a clinical investigation was conducted. The procedures performed in the project did not need the approval of the Institutional Animal Care and Use Committee, which was confirmed by the Local Ethical Committee at the University of Warmia and Mazury in Olsztyn with edict LKE.31.01.2020. Samples were taken longitudinally, not later than 12 h after delivery (time point I) and again after the first (time point II) and second (time point III) months of life (Supplementary Table 1). Samples from the left and right eye were individually collected from the ventral conjunctival fornix using sterile cotton swabs soaked in sterile 0.9% saline (Sarstedt, Copan, Brescia, Italy) (34 (link)). Special care was taken not to contaminate the swabs by contact with the eyelids or eyelashes. The left and right eye of each foal was swabbed once and the procedure was duplicated. A total of 24 swabs (4 per animal) were collected during each sampling. Specimens were quickly placed in cryogenic tubes at a temperature of −20°C for freezing and storage. After the conjunctival swab collection was completed, samples were shipped in an expanded polystyrene box with a cooler (at a temperature of 2–4°C) by courier directly for analysis (Genomed, Warsaw, Poland). The swabs were not frozen again. The same method of swabbing and storage is commonly used in human medicine to avoid contamination with other microbiota (3 (link)).
Genomic DNA was isolated immediately after delivery of samples using a Genomic Mini AX Bacteria kit (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s instructions, with an additional mechanical lysis of each sample effected by zircon balls in a FastPrep homogeniser (MP Biomedicals, Irvine, CA, USA), using a previously reported procedure (28 (link)). Concentration of DNA was measured through the fluorometric method, using a Qubit 4 fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA). The presence of bacterial DNA was confirmed in a qPCR reaction, using 1055F (5′-ATGGCTGTCGTCAGCT-3′) and 1392R (5′-ACGGGCGGTGTGTAC-3′) universal primers for 16S rRNA (5 (link)) and demineralised water as a negative control. The metagenomic analysis of bacteria and Archaea was based on the amplification of the V3–V4 hypervariable region (encompassing approximately 469 base pairs) of the 16S rRNA gene. For the amplification of the selected region and the preparation of DNA libraries, a pair of 341F and 785R primers and NEBNext Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) were used. For the measurement of DNA concentration, 1μL of the reaction mixture was taken immediately after the PCR. The unpurified product contained the reaction mixture with primers and primer dimers; therefore, the result for the negative control was not zero. Concentrations up to a value of approximately 1.5 ng/μL were considered acceptable. When the negative control was above this value (indicative of contamination with PCR reagents) the reaction was repeated. Arbitrarily, it was assumed that the lowest DNA concentration for the testing sample should be twice the minimal concentration of the negative control. Finally, purification of samples was performed with AMPure XP (Beckman Coulter, Indianapolis, IN, USA), followed by final DNA measurement by the fluorometric method on a Tecan reader (Männedorf, Switzerland). A PCR was carried out to index DNA in 50 μL reaction volumes. Next-generation sequencing (NGS) was performed on a MiSeq sequencer (Illumina, San Diego, CA, USA) in paired-end technology (PE300) by Genomed (Warsaw, Poland). MiSeq Reporter v.2.6 software (Illumina) was used for data analysis. To ensure the classification of reads at the species level, bioinformatic analysis was carried out with QIIME software (1 (link)), a semiquantitative approach to microbial ecology based on the SILVA v.138 database of reference sequences (24 (link)). Data analysis was performed of all sequences obtained from NGS and filtered. The diversity of the foals’ microbiota was analysed using alpha (Shannon and Simpson) and beta (Bray–Curtis) diversity indices (9 (link)). The diversity of microbial communities was compared in two ways: in all individuals between samplings and as a mean for the group between samplings, using sequence reads. All diversity indices were calculated for all six foals. Paired one-sided t-tests were used to verify statistically significant changes in biodiversity between collected samples. The calculations were made separately at the family, genus and species taxonomic levels. All calculations were performed in the MATLAB R2020a environment (MathLabs, Natick, MA, USA). Taxa present in amounts equal to or greater than 1% of the total identified DNA sequences at least in one of the individuals were classified as abundant, as proposed by Kim et al. (11 (link)). If the proportion of identified sequences was <1%, taxa were classified as nonabundant.
