Avian sperm-borne RNAs: optimisation of a new isolation protocol

ABSTRACT 1. Sperm-borne RNAs are involved in sperm and embryonic protein translation, the regulation of early development and the epigenetic inheritance of the paternal phenotype. Sperm-borne RNA purification protocols generally include a cell purification stage to discard contamination by somatic cells. In avian species, no protocol is currently available to isolate all the populations composing sperm-borne RNAs. 2. This study evaluated the presence of somatic cells in semen samples of chickens and quails using visual examination after fluorescent nuclei staining. The efficiency of somatic cell lysis buffer (SCLB) on chicken liver cells and its impacts on chicken sperm cell integrity was explored. Three different approaches were tested to isolate RNA: two developed for mammalian sperm cells and a commercial kit for somatic cells. The efficiency and reliability of each approach was determined based on RNA quality and purity. Eventually, the presence of miRNA and mRNA in purified avian sperm-borne RNAs was investigated by RT-(q)PCR. 3. No somatic cells were found in chicken and quail semen. The SCLB totally lysed chicken liver cells but also induced sperm cell necrosis. Consequently, this treatment wasn’t performed on samples prior to RNA isolation. Among the tested RNA purification protocols, the commercial one was the least variable and isolated RNA with the highest purity levels. No DNA contamination was observed. Furthermore, the samples contained miRNA and mRNA already known as present in mammalian sperm cells (gga-miR-100-5p, gga-miR-191-5p, GAPDH and PLCZ1), but mRNAs associated with leucocytes (CD4) and Sertoli cells (SOX4, CLDN11) were not detected. This protocol was successfully applied to quail sperm cells. 4. Altogether, the study reveals that it is unnecessary to pre-treat samples to remove somatic cell contamination before RNA purification and successfully describes an isolation protocol for sperm-borne RNAs, including small non-coding and long coding RNAs, in two distinct avian species highly valuable as biological models.


Introduction
In addition to being one of the most important protein sources for human consumption in the world, the chicken and the quail are two avian species highly valuable as scientific models in embryology, behaviour and genetic and genomic studies (Sellier et al. 2006;Parker and McDaniel 2009;Archer and Mench 2017;Serralbo et al. 2020).More recently, these species have been used to decipher transgenerational epigenetic inheritance mechanisms (Leroux et al. 2017;Guerrero-Bosagna et al. 2018), whereas little is currently known about the general epigenetic mechanisms in avian species (Frésard et al. 2014;David et al. 2017David et al. , 2019)), especially in germ cells such as sperm cells (Shafeeque 2014).
As components of the epigenetic machinery, small noncoding RNAs (sncRNAs), especially miRNA and tsRNA, seem to play a role in the regulation of early development and in the epigenetic inheritance of the paternal phenotype to the offspring (Chen et al. 2016;Schuster et al. 2016;Jodar 2019;Blévec et al. 2020;Cecere 2021;Chan et al. 2020;Wu et al. 2020) and may be used as fertility biomarkers (Abu-Halima et al. 2013;Fagerlind et al. 2015;Shangguan et al. 2020).These RNA populations are a part of the highly diverse sperm-borne RNAs, composed of small and long (non-)coding RNAs (Schuster et al. 2016).Whereas multiple studies aimed to describe and understand the role and the nature of mammalian spermborne RNAs (Chen et al. 2016;Schuster et al. 2016;Jodar 2019;Blévec et al. 2020;Chan et al. 2020;Wu et al. 2020), only two studies attempted to extract and explore the mRNA transcriptome of chicken sperm cells (Shafeeque et al. 2014;Singh et al. 2016), without including all the other RNA populations.
Experiments performed on mammalian species revealed the necessity of specific sperm-borne RNA isolation protocols, according to species, due to differences in sperm morphology and chromatin condensation (Das et al. 2010;Pantano et al. 2015;Sellem et al. 2020;Sahoo et al. 2021).Furthermore, these studies suggested certain semen pretreatment before RNA isolation to purify sperm cells by removing any somatic cell contamination (Pantano et al. 2015;Capra et al. 2017;Alves et al. 2020;Sahoo et al. 2021).
The following study developed a complete procedure for RNA isolation, including sncRNAs and coding RNAs, from sperm cells from two different avian species -the chicken and the quail.After controlling sperm cell purity in fresh semen, three different protocols were applied to the samples to isolate sperm-borne RNA, and efficiency was evaluated in terms of RNA purity and patterns as well as the presence of miRNA and mRNA.

Ethics approval
All experiments were carried out in accordance with the European welfare, and procedures were approved by the French Ministry of Education and Research (APAFIS #-34415-202112141205965 and #4026-2016021015509521).

Animals
Adult T44 roosters (Gallus gallus domesticus, Hendrix Genetics -Sasso, Sabres, France) and Robin male quail (Cortunix japonica, Robin, Maché, France) were housed in individual cages at the PEAT INRAe Poultry Experimental F a c i l i t y ( 2 0 1 8 , h t t p s : / / d o i .o r g / 1 0 . 1 5 4 5 4 / 1 .5572326250887292E12).They were maintained under a lighting regimen of 14 h light:10 h darkness cycle, at 21°C controlled temperature and fed a standard commercial diet and water ad libitum.Roosters used for semen pre-treatment before RNA extraction were 30-34 weeks old (eight roosters) and 38 weeks for testis sampling.The birds used for the development of sperm-borne RNA extraction protocol were 42 weeks old (five birds).The adult male quails used in all the experiments were 23-25 weeks old (nine birds).All individuals had a fertility rate >80%.

Tissue sampling
Semen was collected individually in 200 µl of BPSE extender for chickens (Sexton 1977) and in 20 µl of TM extender for quails (Thélie et al. 2019), by dorso-abdominal massage as previously described (Burrows and Quinn 1937).Care was taken to avoid any contamination with transparent fluid and other cloacal products, such as foam from the quail.
After slaughtering, testes from two chickens at 38 weeks old and two quail at 25 weeks old were excised and immediately frozen in liquid nitrogen and stored at −80°C.Only left testes were used for further experiments.

Evaluation of the presence of somatic cells in avian semen
The presence of somatic cells in chicken and quail semen was explored by visual examination after fluorescent nuclei staining.Briefly, 4 µl of semen per sample was fixed in 100 µl of Antigenfix (Diapath, Voisins-le-Bretonneux, France) for 5 min at room temperature.10 µl of fixed sperm cell solution was placed onto a Superfrost slide (Fisher Scientific, Pittsburgh, Pennsylvania, USA) and dried.Cell nuclei were revealed by Fluoroshield with DAPI (F6057, Sigma-Aldrich, Saint-Quentin-Fallavier, France).Visual examination was performed using a microscope (counting 10 fields of approximately 3,000 sperm cells/sample) to evaluate the proportion of somatic cells (nuclei with round shape) and sperm cells (long and arrow nuclei).Visual observation was performed on an Axioplan 2 fluorescent microscope (Zeiss, Rueil Malmaison, France), and images were acquired with an Infinity 3 camera (Lumenera, Ottawa, Ontario, Canada).

Somatic cell lysis from chickens
Two solutions were used to lyse chicken somatic cells by osmotic stress: water and Somatic Cell Lysis Buffer (SCLB) (Pantano et al. 2015).The efficiency of each solution was first tested on chicken hepatocellular carcinoma epithelial cell lines (LMH cell line, donation of C. Praud, INRAE-BOA).Briefly, the cells were thawed at 37°C for 1 min, washed twice in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, Saint-Quentin-Fallavier, France) and resuspended in 1 mL of DMEM.Then, 50 µl of washed LMH cells was kept in 500 µl of DMEM or subjected to 500 µl of water or SCLB, freshly prepared, for 5 min at 4°C (n = 3 per condition).The cells were centrifuged 5 min at 1000 × g to remove lysis solutions and then resuspended in Lake 7.1 extender (Lake and Ravie 1981).Cell recovery after treatment was evaluated by flow cytometry (EasyCyte Guava, IMV Technologies, L'Aigle, France), after SYBR-14/Propidium iodide fluorescent dye staining (Molecular Probes™ LIVE/ DEAD™ SYBR-14/propidium iodide (PI) fluorescent dyes, L7011, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), as previously described for chicken sperm cells (Thelie et al. 2019).The proportion was determined on forward-and side-scatters on a total of 15 000 events per sample.A similar experiment was performed on chicken sperm cells (2.5 × 10 8 sperm cells per animal per experimental point) in a final volume of 500 µl of Lake 7.1 or 500 µl SCLB.
In addition to sperm cell recovery, sperm cell integrity was determined as the percentage of intact sperm cells (stained by SYBR-14) compared to the total number of sperm cells (stained by SYBR-14 or by SYBR-14 and PI).

Sperm-borne RNA purification
Chicken semen (n = 5), without SLCB treatment, was divided into three equal samples after being washed by PBS 1× (P4417, Sigma-Aldrich, Saint-Quentin-Fallavier, France).Three RNA purification protocols were used, one based on Trizol extraction and already developed in pigs (Gòdia et al. 2018), the other developed in cattle based on βmercaptoethanol/RLT buffer/Trizol (Sellem et al. 2020) and the Qiagen miRNeasy Mini Kit (217004, Qiagen, Courtaboeuf, France).Protocols were performed based on the published literature.RNA extraction was based on the Qiagen miRNeasy Mini Kit protocol.Briefly, sperm cells were homogenised in 700 µl of Qiazol Lysis reagent by Ultra-Turrax T 25 (IKA, Staufen, Germany) for about 30 s and incubated at 30°C for 20 min.Afterwards, the remainder of the Qiagen miRNA purification protocol was performed according to manufacturer's recommendations.The RNA purity and concentration were assayed using Nanodrop ND-1000 UV-Vis Spectrophotometer (Marshall Scientific, New Hampshire, USA), using Qubit RNA HS Assay Kits (Q32852 Molecular Probes, Thermo Fisher Scientific, Waltham, Massachusetts, USA) on a Qubit Fluorometer 4.0 (Molecular Probes by Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts, USA), according to the manufacturer's instructions.The integrity of RNA was verified using an Agilent 2100 bioanalyzer with RNA Pico kit (Agilent Technologies, Les Ulis, France), according to the manufacturer's instructions.
The RNA extraction from testes (chickens and quail, two per species) and from quail semen (nine birds) was performed, as previously described, using a Qiagen miRNeasy Mini Kit (Qiagen, Courtaboeuf, France).Furthermore, in order to better evaluate the efficiency of Qiagen protocol to isolate sperm-borne RNA from a large range of avian sperm cell quantities, a standard curve was derived from one unique chicken semen sample with five serial dilutions (dilution factor: twofold).Sperm-borne RNA from each sperm quantity (two replicates) was isolated and quantified as previously described.

mRNA and miRNA analysis by RT-(q)PCR
Retrotranscription (RT) to investigate the presence of mRNA and miRNA in the purified sperm-borne RNA was, respectively, performed from 100 and 20 ng of total RNA, using Superscript II (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and hexamer random primers (Invitrogen) or using miRcury RT enzyme (Qiagen, Courtaboeuf, France), respectively, following manufacturer's instructions.Negative control samples were included by performing the RT reaction without the Superscript II and RNA matrix.The presence of mRNA was revealed using Platinum Taq DNA polymerase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA).Briefly, after a denaturation step of 94°C for 2 min, 35 cycles of 30 s at 94°C, 60 s at 64°C and 1 min at 72°C were applied.The miRNA expression was explored using miRcury SYBR polymerase (Qiagen, Courtaboeuf, France) and CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Marnesla-Coquette, France), allowing real time and classical PCR.Primer sequences for mRNA were designed using NCBI Primer blast software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and are shown in Table 1.Primers for miRNA (gga-miR-100-5p and gga-miR-191-5p) were derived from Qiagen Gene Globe (Qiagen, Courtaboeuf, France).
To assess the presence and specificity of the amplification, every PCR product was checked on a 2% agarose gel, and those showing mRNA expression were sequenced by Sanger sequencing (Eurofins Genomics, Ebersberg Germany).To check for contamination, DNA was extracted from chicken sperm cells, as previously described (Anvar et al. 2015).
To determine the efficiency of purification protocols, two samples of purified sperm-borne RNA (3 µL of 1:59 RT dilution for each qPCR point) were submitted for qPCR to evaluate the quantity of purified miRNA obtained by the tested protocols.The Cq (quantification cycle) was determined as the average of the two replicates and miRNA expression obtained by each purification protocol was calculated by comparison to the one obtained with Qiagen protocol as reference:

Statistical analyses
All statistical analyses were performed with R software, version 4.0.5 (R Core Team 2017).The impact of somatic lysis procedures on LMH somatic cells and RNA purity according to extraction procedures were explored by Kruskal-Wallis test followed by Dunn's Test to reveal significant differences between the two treatment conditions.The impact of SCLB on chicken sperm cells was investigated by paired Wilcoxon test.A P value ≤0.05 was considered as indicating significant differences between the two conditions.

Evaluation of somatic cells in avian semen
The presence of somatic cells in avian semen was investigated visually under fluorescent microscope after nuclei staining to discriminate nuclei of somatic and sperm cells.No somatic cells were observed in chicken semen, whereas some sperm cell aggregates were revealed (Figure 1

Impact of SCLB on chicken somatic and sperm cells
In order to find the best conditions to remove somatic cell contamination from avian semen, two solutions (water and SCLB) were tested to cause osmotic shock in chicken somatic cells (LMH cells, Figure 2).Although no significant impact was observed with water, SCLB induced a total lysis of chicken somatic cells (more than 99% reduction) (Figure 2(a)).However, when applied to sperm cells, SCLB caused a significant reduction in sperm cells (about 75% reduction, Figure 2(b)) and a large increase in sperm cells with damaged membranes (more than 18-fold, Figure 2(c)).

RNA purity obtained from avian sperm cells
In order to determine the best protocol for purification of sperm-borne RNAs, three different protocols were selected, two which were already used in pigs (Trizol; Gòdia et al. 2018) and in cattle (β-mercaptoethanol/RLT buffer/Trizol -  BME+RLT+Trizol; Sellem et al. 2020) and one commercially developed for somatic cells (Qiagen miRNeasy Mini Kit) (Figure 3).Whereas all procedures succeeded in purifying sperm-borne RNAs with typical pattern (i.e.presence of high amount of small RNAs -Figure 3(a)), only the Qiagen protocol revealed a consistent pattern between samples (Figure 3(a) and Supplementary Figure S1).Furthermore, the analysis of protein contamination in RNA samples revealed the highest purity of RNA extracted with the Qiagen miRNeasy kit (Figure 3(b)).
The RNA integrity number (RIN) of RNA samples obtained from chicken sperm cells with the Qiagen miRNeasy kit was between 1.3 and 2.6 with an average of 2.26.When applied to quail sperm cells, the Qiagen miRNeasy kit led to similar sperm-borne RNA patterns and RIN as the ones obtained in chickens (RIN between 1.5 and 3.5 with an average of 2.55 -Figure 3(a)).

mRNA and miRNA presence in avian sperm-borne RNA
RT-PCR experiments revealed the presence of specific mRNAs and miRNAs in sperm-borne RNAs purified from chicken sperm cells (Figure 4).Indeed, whereas no mRNA relative to lymphocytes or Sertoli cells were detected (CD4 and CLDN11/SOX9 mRNAs respectively), chicken sperm cells expressed PLCZ1, which is known to be specific to gonadal cells, the ubiquitous mRNA GAPDH (Figure 4(a)) and two miRNAs, previously demonstrated as expressed in bovine sperm cells Figure 4(b); Sellem et al. 2020).
All these mRNAs and miRNAs were investigated in chicken testes and their presence was confirmed.Furthermore, the CD4 primers were designed to discriminate PCR products obtained from DNA or RNA matrix, based on different product sizes (Table 1).However, PCR products obtained from chicken sperm cell DNA only corresponded to one band of ~300 bp, but those obtained from RNA extracts presented one specific band at ~216 bp, exhibiting the absence of DNA contamination in these samples (Figure 4(a)).

Sperm-borne RNA purification efficiency
In order to evaluate the efficiency of purification protocols, RT-qPCR experiments were performed using the same amount of sperm-borne RNA obtained with each tested technique to determine the quantity of isolated miRNA (Figure 5).For the two investigated miRNAs (gga-miR-100-5p and gga-miR-191-5p), the expression obtained with the Qiagen protocol was more than 12-fold higher compared to the other tested protocols (Figure 5).Using serial dilutions of one unique chicken semen sample (Figure 6), the Qiagen protocol was able to efficiently isolate sperm-borne RNA from 5 × 10 7 to 10 9 sperm cells, leading to about 30 and 1760 ng of total RNA, respectively.This experiment established that the mean quantity of total RNA in a single chicken sperm cell was about 1.7 fg.

Discussion
More and more studies have reported the importance of sperm-borne RNAs in sperm biology, such as the production of new proteins (Zhao et al. 2009), as well as the regulation of embryonic early development (Jodar 2019) and the transmission of paternal phenotype (Chan et al. 2020).In mammalian species, somatic cells may contaminate semen samples.For instance, mouse semen contains about 4-5% of the somatic cells (Pearson et al. 2018).Consequently, in addition to a visual determination of this kind of contamination as performed in cattle (Sellem et al. 2020), a large amount of studies aiming to purify sperm cells from semen samples avoid somatic cell contamination with two major approaches; gradient density centrifugation using Percoll to select only motile sperm cells (Capra et al. 2017;Alves et al. 2020), and osmotic stress specifically targeting somatic cells with SCLB (Pantano et al. 2015;Sahoo et al. 2021).Both approaches are well-founded, depending on the purpose of the study, whereby density gradient centrifugation using Percoll induces an important bias by selecting only the most motile sperm cells, discarding somatic cells and lessmotile sperm cells, and thus focusing on a specific part of the cells constituting the semen.However, these lessmotile cells may interact with the female epithelia and induce physiological responses (Mahé et al. 2021).Consequently, RNA purification obtained from semen treated by Percoll centrifugation may not reflect all the complexity of semen components and quality, which suggests that semen treatment by SCLB provides an interesting approach to specifically remove somatic cells without impairing sperm cells.1).At the difference of other primer pairs, the ones designed for CD4 discriminated products from RNA and DNA matrix.Some PCR products were investigated as signatures of lymphocyte (CD4), Sertoli (CLDN11 and SOX9) or gonadic cells (PLCZ1) whereas GAPDH was used as control from its ubiquitous presence in all cell types.(b) The presence of miRNA in chicken sperm-borne RNAs was determined by the obtention of a PCR product for two miRnas known as expressed in mammalian gonadic cells: gga-miR-100-5p and gga-miR-191-5p.Spz= sperm cells.Neg.= Negative samples as RT reaction without RT enzyme.
The presence of somatic cells has been reported in chicken epididymis lumen, which suggested potential contamination of ejaculated semen by somatic cells, but this may be an artefact of tissue-handling and treatment (Tingari and Lake 1972).Confirming previous reports (Santiago 2015;Villaverde-Morcillo et al. 2015), the current data revealed the absence of somatic cells in ejaculated semen from adult and mature chickens or quails (Figure 1).The presence of somatic cells in semen may depend on bird age, breed, season and environmental conditions as well as initial fertility status (Shanmugam et al. 2014;Santiago-Moreno et al. 2018;Tesfay et al. 2020;Du et al. 2021;Shi et al. 2021), resulting from spermatogenesis or post-gonadic maturation impairments.Consequently, it was necessary to check somatic cell absence in all semen samples used for sperm-borne RNA isolation by visual examination before treatment.
To avoid any putative somatic cell contamination (undetected by visual examination), the study explored the impact of SCLB on chicken sperm cells.Compared to water, SCLB treatment induced the total destruction of LMH cells (Figure 2(a)), confirming its efficiency in lysing chicken somatic cells.When applied to chicken sperm cells, SCLB caused a reduction in sperm cell numbers and an increase in sperm cells with membrane breaks (Figure 2(b,c)), which suggested the induction of chicken sperm cell necrosis.In dendritic cells, necrosis is associated with cellular RNA content release (Karikó et al. 2004;Brentano et al. 2005), and a similar phenomenon may occur in sperm cells.The SLCB treatment of sperm cells before RNA isolation may induce RNA release from injured sperm cells with membrane breaks.Consequently, all sperm-borne RNA populations may not be represented in SLCB treated samples, leading to an important bias to finely explore these molecular components.Although this negative impact of SCLB on sperm cell integrity has been reported in mice (Pearson et al. 2018), no investigation has been undertaken in other animal species where this treatment is applied (Pantano et al. 2015;Sahoo et al. 2021).Thus, new sperm cell purification methods, reflecting total sperm cell complexity within a semen sample, remain to be developed to avoid somatic cell contamination for further RNA investigation, but without impacting sperm cell integrity.
Considering the absence of somatic cells in semen and the negative impact of SCLB treatment on sperm cells, the semen pre-treatment before RNA isolation appears unnecessary in chickens and quails.Consequently, three different protocols developed for isolating all RNA populations (small and long, coding and non-coding) were applied directly on chicken  semen without prior SCLB treatment.Only the Qiagen miRNeasy kit derived consistent RNA patterns between semen samples, with a high amount of small RNA compared to long RNA and lower protein contamination (Figure 3 and Supplementary Figure S1) as well as higher efficiency of isolated miRNA (Figure 5).Interestingly, the RNA patterns obtained in this study were different from others reported (Shafeeque et al. 2014), although both applied protocols did not involve RNA size selection.Indeed, whereas Shafeeque et al. (2014) reported no small RNA signals but a large majority of long RNA, the current data revealed another RNA pattern where small RNA appeared predominant (Figure 3(a)).Even if Trizol and BME+RTL+Trizol protocols were not consistent, when a sperm-borne RNA pattern was obtained, it was close to those obtained with the Qiagen protocol.Different technical approaches confirmed the higher presence of small RNAs compared to long RNAs in chicken semen.Furthermore, these patterns were similar to the ones obtained in mammalian species (Wu et al. 2020;Pantano et al. 2015), which suggested the Qiagen protocol as the most suitable RNA isolation protocol for obtaining RNAs from chicken sperm cells enriched in small RNAs.The difference of sperm-borne RNA patterns between the current isolation protocol and the one used by Shafeeque et al. (2014) remains to be explored.Consistently, the data showed that the Qiagen miRNeasy protocol can be easily applied to quail sperm cells, revealing similar RNA pattern with high levels of small RNAs and very few long RNAs (Figure 3(a)).
The absence of high 18S and 28S peaks revealed in the Bioanalyser experiments (Figure 3) reinforces the idea of the absence of somatic cell contamination, as previously observed by flow cytometry and visual examination (Figure 1).All these markers were present in testicular extracts, containing blood and Sertoli cells, but were undetectable in chicken sperm cell isolated RNAs, which confirmed the presence of undetectable somatic cells in chicken semen.Furthermore, using primers able to discriminate PCR products from DNA rather than from RNA, tests confirmed the absence of DNA contamination in sperm-borne RNA extracts (Figure 4(a)).
The current study revealed the presence of GAPDH, previously known to be expressed in sperm cells (Sahoo et al. 2021), including in chickens (Shafeeque et al. 2014).Indeed, GAPDH, involved in carbohydrate metabolism, is known as an ubiquitous mRNA, generally used as a reference for somatic cell gene expression analysis (Vitorino Carvalho et al. 2019) and is expressed in sperm cells in goats (Sahoo et al. 2021).Its expression in avian sperm cells is confirmed by this study (Figure 4(a)).The expression of PLCZ1, involved in egg activation was reported (Satouh 2022), in testicular samples and in chicken sperm cell extracts, as previously shown (Shafeeque et al. 2014).In addition to these mRNAs, the data showed the expression of two miRNAs Figure 4(b)), known to be present in bovine sperm cells (Sellem et al. 2020).The presence of mRNAs and miRNAs was controlled in RNA samples purified from quail sperm cells (Suplementary Figure S2), validating the isolation protocol in other avian species.
There was a possibility for using the Qiagen protocol for a large range of sperm cell quantity from 5 × 10 7 to 10 9 sperm cells, with a mean of 1.7 fg of total RNA per sperm cell (Figure 6).This was 5 to 10 times less than the quantity of sperm-borne RNA generally described in mammalian sperm cells (Sahoo et al. 2021) but, due to the large quantity of sperm cells in each chicken semen sample, it was still sufficient for RNA-seq studies (to 300 ng to 1 µg) (Sahoo et al. 2021).
This study is the first attempt to isolate and investigate all the complexity of sperm-borne RNAs in two distinct avian species, highly valuable as biological models -the chicken and the quail.The results showed that SCLB pre-treatment of semen was unnecessary to remove somatic cell contamination, as generally performed in mammals.Nevertheless, in further experimentations, it is necessary to validate the absence of somatic cells in semen samples, at least by visual examination, depending on the age and species.Furthermore, the data confirmed the efficiency and reliability of the Qiagen miRNeasy mini kit protocol to isolate all RNA populations present in avian sperm cells, including miRNAs and mRNAs.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Observation of somatic sperm cells in avian semen.Avian semen samples were subjected to visual observation after DAPI staining (a for chicken semen and b for quail semen).DAPI stained nuclei of somatic cells tend to be large round shapes whereas nuclei of chicken (a) and quail (b) sperm cells are long and arrow.Aggregates of sperm cells are indicated by white arrows.

Figure 4 .
Figure 4. Presence of two different RNA populations (mRNA and miRNA) in chicken sperm cells.The presence of mRNA (a) and miRNA (b) was observed by RT-PCR, followed by electrophoresis migration of PCR products, on a 2% agarose gel.(a) Various mRNA presence was explored using different primer pairs (Table1).At the difference of other primer pairs, the ones designed for CD4 discriminated products from RNA and DNA matrix.Some PCR products were investigated as signatures of lymphocyte (CD4), Sertoli (CLDN11 and SOX9) or gonadic cells (PLCZ1) whereas GAPDH was used as control from its ubiquitous presence in all cell types.(b) The presence of miRNA in chicken sperm-borne RNAs was determined by the obtention of a PCR product for two miRnas known as expressed in mammalian gonadic cells: gga-miR-100-5p and gga-miR-191-5p.Spz= sperm cells.Neg.= Negative samples as RT reaction without RT enzyme.

Figure 5 .
Figure 5. Expression of two miRNA in chicken sperm cells according to sperm-borne RNA purification protocols.miRNA expression (gga-miR-100-5p and gga-miR-191-5p) was evaluated by RT-qPCR in chicken sperm-borne RNA obtained by each tested purification protocol.miRNA expression obtained by each purification protocol was calculated by comparison to the one obtained with Qiagen protocol as reference.Data shown are the mean of two different samples obtained with each protocol.

Figure 6 .
Figure 6.Efficiency of sperm-borne RNA isolation with Qiagen protocol from one unique chicken semen sample.A standard curve was derived from one unique chicken semen sample with five serial dilutions (dilution factor: 2-fold).Sperm-borne RNA from each sperm quantity (2 technical replicates) was isolated with Qiagen protocol and quantified with Qubit technology.

Table 1 .
Primers used for PCR experiments.
F indicates the forward primer and R, the reverse one.