Modified methods for bovine sperm RNA isolation for consistent quality and RNA yield

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. This study aimed to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bull for further transcriptome analysis. Ten fresh ejaculates from each Sahiwal (n = 10 bulls x 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. Swim-up technique was used for live sperm separation and recovery. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) resulted significantly (P < 0.05) higher total RNA quantity (300-340 ng/μL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm. This method will help with the interpretation of data from animal models and the consistency of clinical assessments of male factor fertility employing RNA molecular biomarkers.


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Semen evaluation is an important criterion for successful cryopreservation and artificial 41 insemination. Traditionally, several methods were developed to evaluate semen quality in the 7 139 from 300-400 X 10 6 sperm/mL and the same concentration of sperm was used for the total RNA 140 isolation.

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Somatic cell removal 142 To ensure the purity of the sperm RNA, removal of the somatic cells must be considered 143 during the RNA isolation procedures. A Somatic cell lysis buffer (SCLB) containing 0.1% SDS, 144 0.5% Triton (X-100) was used to lyse somatic cells for sperm cell purification [3,4,25,34,35]. The pre-prepared sperm pellet was mixed with 1 mL of PBS, and washed three times by 158 centrifugation at 3000 rpm for 5 min at 4°C for purification of the sperm cell from other somatic cells, germ cells and leukocytes. According to the manufacturer's instructions, total sperm RNA homogenised 25-30 times with a 20-G needle attached to a 5-mL syringe. After vortexing for 5 163 minutes, the samples were incubated at room temperature (RT) for 5 minutes until the sperm 164 membrane was completely dissociated. 200 µl chloroform was added to the lysate and 165 thoroughly shaken by hand for at least 20 seconds, followed by 5 minutes at room temperature. 166 The mixture was centrifuged at 12,000 X g for 20 min at 4°C to separate the phases. After was added and gently mixed by reversing the tubes. The mixture was kept for 10 min at RT and 171 centrifuged at 12,000g for 15 min at 4 °C. Supernatant was discarded after centrifugation and 1 172 mL of 99.99% ethanol was added to the RNA pellet and again centrifuged at 12,000g for 10 min. 173 Finally, ethanol was removed, and the RNA pellet was air-dried. The pellet was dissolved in 40 174 µl of nuclease-free water, followed by the addition of 2 µl of RNase inhibitor (20 U/l, 175 Invitrogen) and 10 mM dithiothreitol (DTT).

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The prepared pellet was resuspended in 1 mL of PBS, and washed three times by 178 centrifugation at 3000 rpm for 5 min at 4°C to purify the spermatozoa by removing the other 179 somatic cells, germ cells and leukocytes.

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TRIzol reagent was used to extract total RNA from spermatozoa. In this procedure the 181 samples were lysed twice with TRIzol reagent for complete dissociation of sperm membrane. In short, 0.5 mL of ice-cold TRIzol was added to sperm pellet and homogenised for a minimum of seconds, 0.5 mL of fresh, chilled TRIzol was added to the supernatant and vortexed for 1-2 186 minutes, and the rest of the procedure was carried out as described in the TRIzol method.

Combined method
188 RNeasy + TRIzol method with β-mercaptoethanol (BME) 189 The pre-prepared pellet was mixed with 1 mL of PBS, and the sperm sample was washed 190 three times by centrifugation at 3000 rpm for 5 min at 4°C to remove the somatic and other germ 191 cells.

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Here, the total sperm RNA was isolated by combining the conventional and kit (RNeasy) 193 methods described by earlier worker [25] with modifications ( Fig. 1). Briefly, the sperm pellet 194 was homogenised with a 20-G needle for about 25-30 times in 1 mL SCLB containing -β 195 mercaptoethanol (10 µl/mL). The homogenized mixture was vortexed for 1-2 min and incubated 196 at RT for 30 minutes. 0.5 mL of ice-chilled TRI reagent was added to the mixture and again 197 subjected to vortexing for 2 minutes. The mixed sample was then maintained at room 198 temperature for 15 minutes without being disturbed before adding 200 µl cooled chloroform. The 199 contents were mixed vigorously by hand for 20 s and kept at RT for 3 to 5 minutes, followed by 200 centrifugation at 12,000g for 20 minutes at 4 °C. The upper aqueous layer was aspirated into a 201 sterilized 2.0-mL tube, and an equal volume of 100 percent ethanol was pipetted in and mixed 202 thoroughly. The mixture was transferred to a mini-spin column provided and processed as per 203 the manufacturer's protocol. Three times elution was done at room temperature with different volumes (30, 25 and 15 µl) of nuclease-free water. Finally, the three elutes were pooled, and 10 205 mM DTT was added to the pooled RNA, which was stored at -80 o C until further processing.

RNeasy + TRIzol method without β-mercaptoethanol 207
The pre-prepared sperm pellet was resuspended in 1 mL of PBS, and the sperm sample 208 in PBS was centrifuged three times for 5 minutes at 3000 rpm at 4°C to separate the spermatozoa 209 from somatic cells, germ cells, and leukocytes, as described above.

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Total sperm RNA was isolated using a modified combination of conventional and 211 RNeasy methods. BME was not used in this method, unlike the previous one [25].     When the ratio of 28S:18S bands is within 1.8 to 2.0 at 260/280 nm, the RNA sample is 242 considered as high quality.

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The data recorded were analyzed using the SPSS software (version 22). One-way 245 analysis of variance (ANOVA) followed by Tukey's post hoc test to compare the means and 246 determine the significant differences between the groups were used to select the input 247 spermatozoa concentration and the protocol used for RNA isolation based on total RNA yield and quality. The values were presented as means ± SE, and p < 0.05 was considered to be 249 statistically significant.

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A swim-up technique was applied to the freshly ejaculated semen sample to recover the 253 motile spermatozoa. The recovered motile sperm was 300-400 X 10 6 sperm/mL and different  (Table 1 and 2).

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The Agilent 2100 Bio-Analyzer was used for RNA integrity number (RIN) to estimate 283 the purity or integrity of RNA. Based on the peaks, the RNA integrity was measured by RNA 284 integrity number value. RIN values are measured from 1 -10, where RIN Value 1 -5 indicates 285 complete degradation and 5 -7 shows partially degraded RNA and RIN value above 7 indicates 286 high quality RNA. In our study, the RIN value of 2.1 to 9.7 was observed in different samples, 287 indicating the RIN of 9.7, highly intact and 2.1 as highly degraded (Fig.2). Persistence of 18S 288 and/or 28S rRNA showing the isolated RNA integrity (Fig.2). The double band rRNA was 289 visualized by running 2.0% agarose gel electrophoresis (Fig. 3).

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In the present study, we considered the motile and spermatozoa free from other 292 contaminating and somatic cells. The total RNA yield was highest in the modified method 293 (TRIzol + RNeasy + BME), i.e., 39.24 ± 0.41ng/µL, 68.87 ± 1.75ng/µL and 340.72 ± 19.31, ng/µL in 30-40 x 10 6 , 70-80 x 10 6 and 300-400 x 10 6 sperm input concentration, respectively as 295 compared to other isolation methods in Sahiwal bull sperm. It was followed by the TRIzol + 296 RNeasy method, double TRIzol method, TRIzol, and RNeasy (Table 1). Similar trends were 297 found in the Murrah bull sperm with the highest total RNA yield in TRIzol + RNeasy + BME 298 method resulting 51.12 ± 2.16 ng/µL, 72.23 ± 2.65 ng/µL and 318.20 ± 37.60 ng/µL in 30-40 x 299 10 6 , 70-80 x 10 6 and 300-400 x 10 6 sperm input concentration, respectively followed by TRIzol As a major source of bacteria found in soil, bedding, and manure, the bull's preputial 341 orifice is one of the most important sources [48] and to minimize the bacterial load from the 342 ejaculates before collection of the semen; preputial washing was done with normal saline.

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Preputial wash helps yield good quality RNA since the ejaculates collected for RNA extraction

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Even though the overall structure of spermatozoa is similar across species, very 355 combative procedures must be required to disrupt the membrane structure of bovine 356 spermatozoa. By heating the lysis buffer supplied in the Qiagen kit, human sperm RNA samples 357 were able to extract [3,4]. This method was ineffective for bull spermatozoa because extensive 358 cellular debris is deposited at the bottom of the tube after the lysis process, resulting in low total 359 RNA quantity. Indeed, mature spermatozoa are not known to be transnationally active, implying 360 that rRNAs required for ribosome assembly may not be present. 361 Five techniques have been attempted and compared to recommend a suitable method for 362 bovine sperm RNA isolation. When membrane-based approaches combined with TRIzol (TRIzol + BME + RNeasy) were compared to other methods, both the amount and quality of 364 recovered RNA were higher (TRIzol, Double TRIzol and RNeasy). Our study's findings agree 365 with earlier researchers [37] who isolated RNA from Holstein Friesian bulls and reported higher 366 RNA yield in RNeasy + TRIzol method compared with the other methods. Higher concentration 367 (37.39 ± 1.04 ng/µL) was found in the present study from 30-40 million sperm as compared to  In conclusion, our findings suggest that the membrane-based methods with TRIzol and 384 somatic cell lysis buffer, BME or DTT, and for generating good quality and quantity RNA from