All experiments in the current study were approved by the Institutional Animal Care and Use Committee of the Northwest A&F University, Shaanxi, China.
Chemicals and reagents
Unless otherwise stated, all chemicals and reagents used in the present study were purchased from Sigma-Aldrich, China. The R848 (TLR7/8 ligand) was obtained from Novus Biologicals (Littleton, CO, USA).
Semen collection and swim-up test
Semen was collected twice weekly from six Guanzhong dairy goat bucks (2–3 years old) using an artificial vagina at the Shaanxi Aonike Guanzhong Dairy Goat Breeding Farm, Fuping, Shaanxi, China. The ejaculate was transported to the lab, and sperm quality was assessed within 30 min. All semen samples with sperm motility > 75% were mixed to avoid the influence of the individual bucks used in the study. The swim-up test is shown in Fig. 1; it was performed as previously described . The sperm were incubated in a goat semen extender  (3 mL per group; 1 × 108 sperm/mL), with several concentrations of R848 for different time intervals at 37 °C. Next, the percentages of sperm in the upper (1 mL) and lower (1 mL) layers were evaluated (number of sperm in upper or lower layer/total sperm). The separated sperm were used for further analysis.
Sperm motility parameter assessment using the computer-aided sperm analysis (CASA) system
Sperm were incubated in a goat semen extender containing 1 μmol/L R848 at 37 °C for 30 min. Then, aliquots (50 μL) of each treated semen sample were diluted with 50 μL of base extender in PCR tubes. After the mixed samples (100 μL) were incubated in a water bath (37 °C) for 5 min, each semen sample (10 μL) was transferred to a pre-warmed glass slide (37 °C) containing a coverslip (CELL-VU® DRM-600, Millennium Sciences lnc. New York, USA). Sperm motility parameters were evaluated using a computer-aided sperm analysis (CASA) system (HVIEW-SSAV8.0, FuZhouHongShiYe Software Technology Co., Ltd., China) . The standard parameters were set at 30 frames/s and sperm tracks were captured at 60 Hz. The measured sperm motility parameters included sperm motility, average path velocity (VAP), average straight-line velocity (VSL), and average curvilinear velocity (VCL). We defined sperm motility as the percentage of sperm with straightness of path (STR) ≥ 75% and VSL > 25 μm/s. A minimum of 500 sperm were observed from at least five randomly selected fields using a phase-contrast microscope (Nikon 80i; Nikon, Tokyo, Japan). All experiments were replicated five times, and performed between July and November 2020.
Immunofluorescence (IF) of testis and epididymis
Three Guanzhong dairy goat bucks (8 months old) were euthanised after overnight fasting with neck vein injections of pentobarbital (50 mg/kg body weight; Release; WDT, Garbsen, Germany) and xylazine (0.5 mg/kg body weight; Xylosol; Ogris Pharme, Wels, Austria) . Then, their testes and epididymis were collected and fixed in 4% (w/v) paraformaldehyde overnight and embedded in paraffin, as described previously [13, 20]; next, the tissue sections of the testis and epididymis were probed with an anti-TLR7 antibody (bs-6601; Bioss Antibodies, Woburn, MA, USA) and anti-TLR8 antibody (ab180610; Abcam, Cambridge, MA, USA) diluted at a ratio of 1:200. After washing with PBS, the antigens (TLR7 and TLR8) were visualised using Cy3-conjugated goat anti-rabbit IgG (ab6939; Abcam), 30 μL fluorescein isothiocyanate-peanut agglutinin (FITC-PNA, 100 μg/mL in PBS), a marker of acrosome in round spermatid and sperm , and 1 μg/mL DAPI, which was used to stain the nuclei. Stained sections were monitored and photographed using an epifluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). For the testes, three cross-sections of seminiferous tubules with more than 100 round spermatids in each tubule were randomly selected using a phase-contrast microscope (Nikon Eclipse C1). Round spermatids of goat testes were stained with TLR7/8 (red) and PNA (green), and the proportion of TLR7/8-positive cells (%) was calculated as follows: (the number of PAN+ and TLR7+ cells/number of PAN+ positive cells) ×100. Consistently, three cross-sections of epididymis tissues with more than 300 sperm in each lumen were randomly selected using a phase-contrast microscope (Nikon Eclipse C1). Sperm of the epididymis tissues were stained with anti-TLR7/8 (red) and PNA (green), and the proportion of TLR7/8-positive sperm (%) was calculated as follows: (the amount of PAN+ and TLR7+ sperm/PAN+ sperm) ×100. Visualisations were performed using a DAPI ultraviolet excitation wavelength of 330–380 nm, with an emission wavelength of 420 nm (blue light emission); an FITC excitation wavelength of 465–495 nm with an emission wavelength of 515–555 nm (green light emission); and a Cy3 excitation wavelength of 510–560 with an emission wavelength of 590 nm (red light emission). All experiments were completed in triplicate from August–December 2019.
Immunofluorescence of sperm
The sperm samples obtained from the dairy goat bucks was spread over slides, air-dried, and fixed in 100% methanol for 10 min. After air-drying, the sample slides were rinsed with PBS three times for 5 min (each time). The sperm samples were then permeabilised with 0.5% (v/v) Triton X-100/PBS at room temperature for 30 min. The sample slide was rinsed with PBS three times for 5 min (each time). The sperm were incubated with a blocking solution (5% BSA) at 37 °C for 30 min. Then, they were probed with anti-TLR7 or anti-TLR8 antibodies at a ratio of 1:100 and incubated overnight at 4 °C. After washing with PBS, the antigens were visualised with Cy3-conjugated goat anti-rabbit IgG (1:2,000; ab6939, Abcam) and 1 μg/mL DAPI. Sperm staining was monitored and photographed using an epifluorescence microscope (Nikon Eclipse C1). The experiments were completed in triplicate from September–December 2019 and July–November 2020.
Analysis of sperm membrane integrity
The membrane integrity of the upper- and lower-layer sperm was evaluated after incubation in an extender containing 1 μmol/L R848 at 37 °C for 30 min. Sperm membrane integrity was evaluated using SYBR-14/propidium iodide (PI), according to a previously described procedure . Briefly, the sperm samples (100 μL) were stained using 0.1 μL of SYBR-14 working solution (100 μmol/L in DMSO) for 10 min at 37 °C, and then using 0.5 μL of propidium iodide (PI) working solution (2.4 mol/L in water) for 10 min at 37 °C. Sperm staining was monitored and photographed using an epifluorescence microscope (Nikon Eclipse C1). More than 200 spermatozoa were assessed per slide. Five replicates of each semen sample were evaluated by the same observer. All experiments were completed in September 2020.
Analysis of sperm acrosome integrity
The acrosome integrity of the upper- and lower-layer sperm was evaluated after incubation in an extender containing 1 μmol/L R848 at 37 °C for 30 min. In accordance with the method described by Ren et al. (2019) , acrosome integrity was measured using fluorescein isothiocyanate-peanut agglutinin (PNA-FITC) staining. Briefly, sperm samples (30 μL) were spread over slides, air-dried, and fixed in 100% methanol for 10 min. The fixed sperm were then stained using 30 μL of PNA-FITC (100 μg/mL in PBS), incubated at 37 °C for 30 min, and rinsed thrice with PBS. Nuclear staining was performed using 1 μmol/L DAPI. The surface dye was washed three times using PBS and air-dried in the dark. The spermatozoa staining was monitored, and the stained spermatozoa were photographed using an epifluorescence microscope (Nikon Eclipse C1). More than 100 spermatozoa were assessed per field, and five randomly selected fields were assessed for each sample. Five replicates of each semen sample were evaluated by the same observer. The experiments were completed in September 2020.
Analysis of sperm mitochondrial activity and ATP levels
The mitochondrial activity changes in the sperm were evaluated using a JC-1 lipophilic cation (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer’s instructions, as described by Li et al. (2020) . Briefly, sperm samples (2 × 106/mL) were stained with 28 μL of JC-1 (stock solution) in PBS (final volume, 100 μL). After incubation at 37 °C for 30 min in the dark, the samples were centrifuged at 600 g for 5 min at 4 °C and washed with JC-1 buffer twice. After resuspension in JC-1 buffer, the sperm samples were immediately analysed using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA), with excitation wavelengths of 490 nm and 525 nm and emission wavelengths of 530 and 590 nm for the JC-1 monomer and the JC-1 polymer, respectively. Sperm mitochondrial activity was calculated using the following equation:
Sperm mitochondrial activity (%) = the value of JC-1 polymer/(JC-1 monomer + JC-1 polymer) ×100%.
Three replicates were evaluated for each sperm sample.
ATP levels of dairy goat sperm were assessed using an ATP Assay Kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions, and as described by Li et al. (2020) . Briefly, 500 μL of semen (8 × 107 sperm/mL) was centrifuged at 4 °C and resuspended in ATP assay lysate (200 μL) to release the intracellular ATP. The sperm sample was then centrifuged at 12,000 g at 4 °C for 5 min and the supernatant was aspirated. The ATP standard solution (0.5 mmol/L) was diluted to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L in succession using ATP assay lysate. The supernatants (50 μL) and standards (50 μL) were added to a 100-μL ATP detection working solution in opaque 96-well plates. The fluorescence intensity of the samples was detected using a multi-detection microplate reader (Bio Tek Synergy H1; Bio Tek, Winooski, VT, USA). Three replicates were evaluated for each sperm sample. All experiments were completed between July and November 2020.
In vitro fertilisation (IVF) using the lower layer TLR7/8-activated sperm
Goat ovaries were obtained from a local abattoir and transported to the laboratory within 4 h in 0.9% sodium chloride solution at 38.5 °C. After removing the connective tissues, fat mass, and fallopian tubes, the ovaries were soaked in 75% alcohol for 30 s and washed 3 times with a 0.9% sodium chloride solution preheated to 38.5 °C. The ovaries were placed into a 60-mm petri dish containing TCM-199 medium  with 6 mg/mL BSA (washing medium) and fixed with tweezers. Subsequently, the follicles on the surface of the ovary were lacerated with a scalpel. The follicles were washed with the oocyte collection fluid to ensure the complete flow of the follicular fluid. The cumulus-oocyte complexes (COCs) were collected under a stereo microscope (LECIA M165 FC; Lecia; German) using a self-made mouth pipette, and COCs with three layers of cumulus cells were used in this experiment. Thirty to forty COCs were placed in a 200-μL drop of BO-IVM medium (IVF Bioscience, Falmouth, United Kingdom), and incubated for 24 h at 38.5 °C and 5% CO2. TLR7/8-activated sperm (50 μL; 2 × 107 spermatozoa/mL) were added to the 30–40 COCs in a 500-μL six-well dish containing BO-IVF (IVF Bioscience) medium and mineral oil and incubated for 18 h at 38.5 °C and 5% CO2. Subsequently, the unfertilised sperm and cumulus cells were removed from the zygotes using 0.1% hyaluronidase PBS. Next, the zygotes were transferred to BO-IVC medium (IVF Bioscience) for culture. After 7 d, the blastocysts were identified and collected, and their sex was determined using PCR. The experiments were performed in triplicate and were completed between July and November 2020.
Artificial insemination (AI) using the lower layer TLR7/8-activated sperm
A CIDR (CIDR®, EaziBreed; Zoetis, Parsippany-Troy Hills, NJ, USA) device was intravaginally placed in the donor Guanzhong dairy goat does (n = 6) on day 0. On day 14, the goats were injected with FSH (Folltropin-V®; Bioniche Animal Health, Belleville, Canada), starting in the morning. In total, 180 mg of FSH was injected over 3 d (50, 50, 30, 30, 10, and 10 mg) at 12-h intervals. The CIDR device was removed from the does on day 17. After 24 h, the donor goats were intra-cervically inseminated with the lower-layer sperm (incubated with 1 μmol/L R848 at 37 °C for 30 min), as described in our previous report . Briefly, an opener was used to open the vagina and observe the changes to evaluate the oestrus period of the donor goat. The oestrus period was defined by the presence of more viscous secretions in the vagina and a congested, loose, and open cervix. Two inseminations after the oestrus period (12 h apart, 0.5 mL with 5 × 107 spermatozoa each time) were performed and the resulting embryos were collected after 5 d for sex determination using PCR. Transcervical embryo recovery was performed as described by Santos et al. (2020) . The experiments were completed in December 2020.
Sex determination of goat embryos by PCR
Each of the blastocysts was transferred to PBS in 0.01% BSA for washing in vitro, and then individually placed in a 0.2-mL enzyme-free centrifuge tube and dispensed with 2.5 μL of genome extract (1 mg/mL Proteinase K, 0.5% Triton X-100, 50 mmol/L Tris-HCl, pH = 8.0). Single blastocysts were then placed in a Polymerase Chain Reaction (PCR) machine (T100; Bio-Rad; United States) and incubated at 65 °C for 3 h and at 95 °C for 10 min. PCR analyses of single blastocysts were performed with KOD FX Neo (KFX-201, Toyobo Co., Ltd., Life Science Department, Osaka, Japan), according to the manufacturer’s instructions. Primer sets recognising the Y- (SRY) and X-chromosomes (B-ACTIN) were generated and used for the PCRs (Table 1). The following PCR amplification conditions were used: initial denaturation at 95 °C for 5 min and 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 68 °C for 40 s, with a final extension at 68 °C for 5 min. Subsequently, the PCR products were visualised by 2% agarose gel electrophoresis. Each lane represented an embryo, except for the Ma and Fe lanes, which represented the DNA of control male (Ma) and female (Fe) dairy goats, respectively. Female embryos were identified by the production of one band (B-ACTIN, 351 bp) and male embryos, by the production of two bands ((B-ACTIN, 351 bp) and (SRY, 162 bp)). All experiments were completed between July and December 2020.
Determination of X/Y sperm ratio by flow cytometry
The proportions of X- and Y-sperm were determined by flow cytometry, as described by Welch et al. (1999) . Briefly, after incubating the sperm with 1 μmol/L R848 for 30 min at 37 °C, the upper or lower sperm layers were centrifuged at 400 g at 37 °C for 5 min. The sperm samples were then diluted to a concentration of 2 × 108 sperm/mL with 500 μL of goat extender containing 8 μL of Hoechst 33342 (2 mg/mL) and tailed off by ultrasound for 1 s. The samples were incubated at 37 °C for 50 min in the dark, with mixing every 10 min. A high-speed sorting flow cytometer (BD FACSAria™ III Cell Sorter; BD, Franklin Lakes, NJ, USA) was used to analyse the proportions of X- and Y-sperm. The experiments were completed between July and November 2020.
Western blot analyses
As described in our previous study , to obtain total protein lysates, the sperm were lysed on ice for 30 min using a cell lysis buffer containing 250 μL of RIPA and 2.5 μL of PMSF. A BCA assay kit was used to determine total protein concentrations and the protein samples with loading buffer were boiled at 100 °C for 10 min. Protein samples (20 μg of protein in each lane) were electrophoresed on 10% SDS-polyacrylamide gels. The proteins were separated by PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with the indicated primary antibodies. Next, the blots were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used were: phospho-AKT (10176–2-AP; Proteintech Group, Inc., China), phospho-NF-κB (3031 s; Cell Signalling Technology, Boston, MA, USA), phospho-GSK3α/β (9331 s; Cell Signalling Technology), total AKT (ab81283; Abcam), total NF-κB (8242 s; Cell Signalling Technology), and total GSK3α/β (5676 s; Cell Signalling Technology). Finally, after washing with TBST, the blots were incubated with an alkaline phosphatase-conjugated secondary antibody (1:10,000 dilutions in TBST) for 1 h at 37 °C. The reactive proteins were visualised using chemiluminescence (ECL) western blot reagents and quantified using ImageJ software. The levels of the target proteins were normalised to those of anti-alpha tubulin (ab7291; Abcam), which was used as an internal control. The experiments were completed between July and November 2020.
All results were expressed as the mean ± standard error of the mean (SEM). Before statistical analysis, all data were tested for normal distribution and homogeneity of variance. Percentage data were transformed by arc-sin square root transformation to normalise the distributions prior to statistical analysis. Statistical analyses of data from at least three replicates were compared using either Student’s t-test or one-way ANOVA, and multiple comparisons were performed with Duncan’s multiple range test using SPSS version 20 for Windows (SPSS Inc., Chicago, IL, USA) (*, P < 0.05; **, P < 0.01).
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