The authors have declared that no competing interests exist.
‡ These authors also contributed equally to this work.
Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.
Due to reductions in fertility and litter size rates, frozen-thawed (FT) boar semen has only been commercially used on a small scale [
Seminal plasma (SP) has been studied as a method of minimizing/reversing cryopreservation injuries. In addition, ejaculate-specific fractions from boars can be used as an alternative to whole ejaculate [
Although the addition of frozen-thawed boar SP results in an improvement in motility as well as membrane integrity [
An epifluorescence microscopy technique previous described by Andrade et al. [
However, there is a limit to the number of spermatozoa counted by epifluorescence microscopy technique. Indeed, a larger amount of cells can be analyzed in a short time period using flow cytometry, increasing the accuracy of such technique [
Therefore, the aim of this study was to (1) introduce a flow cytometry fourfold stain technique for simultaneously evaluating of plasma, acrosome and mitochondrial membrane excluding cell debris; (2) evaluate these parameters in association with sperm kinetics, tyrosine phosphorylation and
The freezing medium (Botu-Sui® - composed of sugars, amino acids, buffers, 20% egg yolk [v/v], antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide [v/v]) was developed and donated by Biotech-Botucatu- Ltda ⁄ME (Botucatu, SP, Brazil). Beltsville Thawing Solution (BTS) was acquired from IMV Technologies (L'Aigle, France). The hormones used were purchased from MSD Animal Health (New York, EUA—Regumate®), Bioneche Animal Health (Ontario, Canada—Lutropin®- V) and Coopers (Brazil—Novormon®). Fetal bovine serum was purchased from Vitrocell—Embriolife (Campinas, Brazil). Hoechst 33342 and JC-1 fluorescent probes were purchased from Molecular Probes (Eugene, OR, USA). Unless otherwise stated, all other chemicals (propidium iodide [PI],
Semen samples were diluted in Tyrode’s albumin lactate pyruvate (TALP) sperm medium [
Flow cytometry was performed with a BD FACSAria flow cytometer (Becton Dickinson, San Jose, CA, USA) controlled by BD FACSDiva 6.0 software (Becton Dickinson). An argon laser at 488 nm and a near-UV laser at 375 nm simultaneously excited the cells. Quality control of the FACSAria operation was performed before every routine analysis using CST Software (Cytometer Setup and Tracking). When necessary, manual compensation of the sperm flow cytometry analysis was performed with the aid of a positive control for each probe used (
A single sperm-rich fraction was collected from nine boars (n = 9) using the gloved-hand technique. Immediately after collection, the semen samples were diluted in TALP sperm medium pre-warmed to 37°C and adjusted to a final concentration of 25 x 106 spermatozoa/mL using a Neubauer hemocytometer. The semen was evaluated for motility characteristics using a sperm class analyzer (SCA—Microptics® - Barcelona/Spain).
Raw semen was diluted in TALP medium to a final concentration of 5 x 106 spermatozoa/mL and split into two aliquots: one was kept its raw diluted conditions, and the other was subjected to three cycles of flash freezing in liquid nitrogen and slow thawing to induce damage to the acrosomal and plasma membranes and to perturb mitochondrial function. Five treatments were prepared with the following fixed ratios of raw diluted semen to flash frozen semen: 100: 0 (T100), 75: 25 (T75), 50: 50 (T50), 25: 75 (T25) and 0: 100 (T0). After treatment allocation, total motility was evaluated using an SCA (Microptics® - Barcelona/Spain).
The sperm flow cytometry fourfold stain method aimed to incorporate the flow cytometry in the technique previous described for the same staining approach using epifluorescence microscopy [
(A) Side scatter (SSC) x forward scatter (FSC) dot plot showing the “Sperm + cell debris” gate. (B) Hoechst 33342 histogram originating from the “Sperm + cell debris” gate to exclude non-cellular particles by a DNA probe, Hoechst 33342. (C) Propidium iodide histogram based on Hoechst 33342 for evaluating sperm cell membrane integrity. IM gate showing spermatozoa with intact plasma membranes and the DM gate showing damaged sperm plasma membranes. (D) Dot plot from the IM gate for analyzing mitochondrial membrane potential (axis y; JC-1 orange) and acrosome integrity (axis x; PSA-FITC), as represented by each quadrant of expected sperm cells. (E) Dot plot from the DM gate for analyzing mitochondrial membrane potential (axis y; JC-1 orange) and acrosome integrity (axis x; PSA-FITC), as represented by each quadrant of expected sperm cells.
Each treatment aliquot (150 μL with 5 x 106 spermatozoa/mL) was stained with H342 (2.33 μg/ mL), PI (100 μg/ mL), PSA (13.3 μg/ mL) and JC-1 (4.08 μM). Samples were incubated at 37°C for 10 min, diluted in 150 μL TALP medium and analyzed by flow cytometry.
Same samples (single ejaculate collected by gloved-hand technique from 9 cross-bread boars) used to sperm flow cytometer fourfold stain was used to dual and triple stain assays. For dual stain analysis (individual sperm compartment was evaluated–
Stain type | Sperm Compartment Evaluated | Exclusion of Cell debris | Cell category | ||
---|---|---|---|---|---|
Plasma Membrane | Acrosome | Mitochondrial Potential | |||
Dual stain |
+ | − | − | + | IP |
− | + | − | + | IA | |
− | − | + | + | HP | |
Triple stain |
+ | + | − | + | IPIA |
IPRA | |||||
DPIA | |||||
DPRA | |||||
Fourfould stain |
+ | + | + | + | IPIAH |
IPIAL | |||||
IPRAH | |||||
IPRAL | |||||
DPIAH | |||||
DPIAL | |||||
DPRAH | |||||
DPRAL |
IP–integrity of plasma membrane, IA–integrity of acrosome, HP–high mitochondrial membrane potential (Δψm), IPIA–simultaneous integrity of plasma and acrosome membranes, IPRA–simultaneous integrity of plasma membrane and reacted acrosome, DPIA–simultaneous damage of plasma membrane and integrity of acrosome, DPRA–simultaneous damage of plasma and acrosome membranes, IPIAH–simultaneous plasma and acrosome membrane integrity and high Δψm, IPIAL—simultaneous plasma and acrosome acrosomal integrity and low Δψm, IPRAH—simultaneous plasma membrane integrity, reacted acrosome and high Δψm, IPRAL—simultaneous plasma membrane integrity, reacted acrosome and low Δψm, DPIAH—simultaneous damaged plasma membrane, acrosome integrity and high Δψm, DPIAL—simultaneous damaged plasma membrane, acrosome integrity and low Δψm, DPRAH—simultaneous damaged plasma membrane, reacted acrosome and high Δψm, DPRAL—simultaneous damaged plasma membrane, reacted acrosome and low Δψm.
1Dual stain was performed by the association of Hoechst 33342 with propidium iodide (PI for IP analysis), or
2Triple stain was performed by the association of Hoechst 33342 with PI and PSA
3Fourfold stain was performed by the association of Hoechst 33342 with PI, PSA and JC-1.
Four whole sperm-rich fractions were obtained from each of six boars (n = 24). Immediately after collection, the concentration was evaluated, and CASA analyses were performed. After the initial analysis, the semen was distributed into three treatments: a control (CT–without handling of seminal plasma); suspended in autologous seminal plasma after centrifugation (CS) and withdrawn seminal plasma after centrifugation (CW). The CS sediment was suspended in its own seminal plasma after centrifugation (500 x g/10 min). The CW samples were centrifuged (500 x g/10 min), and the supernatant was completely separated from the pellet by aspiration, reserved and processed as described below in “Seminal plasma collection and storage”. The CT, CS and pellet fractions obtained from CW were suspended at ambient temperature (25°C) using a freezing extender containing 2% glycerol and 2% methylformamide [v/v], as a cryoprotectants, to obtain a final concentration of 300 x 106 spermatozoa/mL and stored in 0.5 mL straws (IMV, Laigle, France). The straws were subsequently placed into an automatic freezing system (TK 3000®; TK Tecnologia em Congelação Ltda, Uberaba, Brazil) and cooled at a rate was -0.5°C⁄ min from 25°C to 5°C. The freezing rate was -20°C⁄ min from 5 to -120°C. Subsequently, the straws were immersed in liquid nitrogen at -196°C and stored in goblets within cryogenic tanks. All straws were kept in liquid nitrogen for a minimum of one week before thawing.
Autologous seminal plasma arising from sperm-rich fraction (SP-SRF) was obtained from the same sample collection for cryopreservation. The supernatant previously obtained was centrifuged (2500 x g/ 30 min) and completely separated from the pellet by aspiration. This sample was then vacuum filtered through disposable filters (0.22 μm in diameter; 99150—Filtermax, TPP®; Switzerland) and stored at -80°C for further use.
Two straws per ejaculate and treatment (CT, CS and CW) were thawed in a water bath at 37°C for 30 sec and diluted to a final concentration of 25 x 106 spermatozoa/mL in a freezing extender. Additionally, two straws per ejaculate from the CW treatment were thawed and diluted with freezing extender supplemented with 10% SP-SRF (v:v) [
A sample aliquot (5 μL) was withdrawn at each incubation time point and placed on a pre-warmed cover slide and evaluated by phase-contrast microscopy (Nikon, Modelo Eclipse 80i) with 100x magnification. Five good fields were examine using SCA (Microptics® - Barcelona/Spain) for evaluating the following parameters: total (TM) and progressive (PM) motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and hypermotility (HIPER). The hyperactivated sperm population was evaluated using Edit/Sort in the software, with ALH > 3.5 μm and VCL > 97 μm/s [
Samples were stained and analyzed as described above in the section “Sperm flow cytometry fourfold stain.”
Semen samples were analyzed for the presence of protein tyrosine phosphorylation on the surface of the sperm membrane [
Six sperm-rich fractions were obtained as described above, from each of two of the best boars based on the results of experiment 2 to sperm viability. The insemination dose was prepared using the pooled sperm-rich fraction of those two boars. This assay was performed only with the CT, CW and CWSP treatments and was carried out as described above. The exclusion of CS treatment was possible only because any centrifugation effect was observed in experiment 2 (see ‘
Whole homologous SP-SRF was obtained from the same two boars used for semen cryopreservation. Sperm-rich fractions were collected using the glove-hand technique and evaluated using the CASA system. Samples with total motility ≥ 80% were pooled and centrifuged twice (500 x g/10 min and 2500 x g/ 30 min). The recovered supernatant was vacuum filtered and stored as previously described.
An insemination dose was prepared in 50 mL of BTS (Beltsville Thawing Solution) with 30 x 106 spermatozoa/ mL. Ten straws of CT and CW were thawed in a water bath at 37°C for 30 sec, and 45 mL of BTS pre-warmed to 37°C was slowly added to the thawed semen. The other ten straws of CW were thawed under the same conditions, and 40 mL of BTS plus 10% homologous SP-SRF (v:v) was slowly added [
Thirty-three gilts were fixed-time inseminated (eleven per treatment; n = 33). The females were orally treated with altrenogest (4 mg/ mL; Regumate®) for 18 days. Twenty-four hours after altrenogest withdrawal, 600 UI of eCG (equine chorionic gonadotropin; Novormon®) was administered [
Five days after IUAI, the gilts were slaughtered for reproductive tract collection. This procedure complied with the legal standards and ethics of the Ethics Committee on the use of Animals at the School of Veterinary Medicine and Animal Science of University of São Paulo, which approved this study under protocol 3066/2013. Before electro-stunning, a pipette was introduced into the cervix to prevent urine reflux. After bloodletting, the reproductive tract was withdrawn using a linea alba incision. Each ovary was identified, and the corpus luteum was counted to verify the fertility rate. The oviduct and cranial uterine horn were dissected, separated and flushed with 20 mL of PBS (phosphate buffered saline) containing 1% fetal bovine serum. The lavage was collected in a pre-heated Petri dish and evaluated under 20x magnification. The fertility rate (FR–number of embryos/sum of oocytes and embryos) was calculated after embryo collection from the flushed oviducts.
Flow cytometry application of the sperm fourfold stain using combined Hoechst 33342, propidium iodide, lecithin from
(A) Linear regression between treatments of the sperm population with plasma and acrosomal membrane integrity and high mitochondrial membrane potential (IPIAH). (B) Correlation between sperm IPIAH and total motility.
Graphic representation of all semen treatment dynamics analyzed using three graphs: a histogram that represents the plasma membrane integrity and two dot plots for analyzing mitochondrial membrane potential (axis y) and acrosome integrity (axis x). The dot plot with green stain represents intact plasma sperm membranes (from the IP histogram gate–plasma membrane integrity), and the dot plot with red coloration represents damaged plasma sperm membranes (from the DP histogram gate–damaged plasma membranes). IPIAH–simultaneous plasma and acrosome membrane integrity and high mitochondrial membrane potential (Δψm), IPIAL—simultaneous plasma and acrosome acrosomal integrity and low Δψm, IPRAH—simultaneous plasma membrane integrity, reacted acrosome and high Δψm, IPRAL—simultaneous plasma membrane integrity, reacted acrosome and low Δψm, DPIAH—simultaneous damaged plasma membrane, acrosome integrity and high Δψm, DPIAL—simultaneous damaged plasma membrane, acrosome integrity and low Δψm, DPRAH—simultaneous damaged plasma membrane, reacted acrosome and high Δψm, DPRAL—simultaneous damaged plasma membrane, reacted acrosome and low Δψm.
Sperm assay by dual or triple stain, in the same sample, overrates spermatozoa potentially fertile. It is noted when raw diluted samples (T100) were evaluated by dual stain, and the samples exhibited 76.07 ± 3.83% of plasma membrane integrity, 95.14 ± 0.64% of acrosome integrity and 76.33 ± 3.14% of high mitochondrial membrane potential (
Stain type | Cell category | Percentage (%) of raw diluted semen | ||||
---|---|---|---|---|---|---|
0 | 25 | 50 | 75 | 100 | ||
Dual stain |
IP | 18.69 ± 6.04 c | 21.63 ± 2.27 b, c | 37.60 ± 3.32 b, c | 53.49 ± 4.84 b | 76.07 ± 3.83a |
IA | 39.92 ± 8.90 c | 50.81 ± 8.14 b, c | 62.94 ± 6.10 a, b, c | 76.66 ± 4.72 a, b | 95.14 ± 0.64 a | |
HP | 8.14 ± 3.05 e | 19.65 ± 1.79 d | 34.22 ± 3.15 c | 55.98 ± 2.57 b | 76.33 ± 3.14 a | |
Triple stain |
IPIA | 17.76 ± 6.39 c | 27.3 ± 5.51 b, c | 39.22 ± 4.69 a, b | 52.1 ± 4.8 a | 75.24 ± 3.83 a |
IPRA | 2.72 ± 0.38 a | 2.07 ± 0.37 a, b | 1.87 ± 0.33 a, b, c | 1.38 ± 0.17 b, c | 0.83 ± 0.09 c | |
DPIA | 23.96 ± 4.40 | 23.50 ± 3.75 | 23.71 ± 3.41 | 24.56 ± 3.36 | 19.89 ± 3.58 | |
DPRA | 57.34 ± 9.03 a | 47.14 ± 8.02 a, b | 35.22 ± 6.10 b, c | 21.97 ± 4.70 b, c | 4.04 ± 0.60 c | |
Fourfold stain |
IPIAH | 0.64 ± 0.30 e | 12.38 ± 0.97 d | 23.92 ± 2.27 c | 38.38 ± 2.46 b | 59.17 ± 2.71 a |
IPIAL | 8.59 ± 4.19 | 8.31 ± 1.54 | 16.45 ± 5.10 | 15.15 ± 4.68 | 12.25 ± 3.00 | |
IPRAH | 0.15 ± 0.08 b | 0.23 ± 0.07 b | 0.39 ± 0.07 a, b | 0.55 ± 0.08 a | 0.54 ± 0.08 a | |
IPRAL | 2.21 ± 0.36 a | 1.83 ± 0.34 a | 1.21 ± 0.21 a, b | 0.83 ± 0.15 b | 0.29 ± 0.06 b | |
DPIAH | 2.29 ± 0.66 | 2.42 ± 0.51 | 2.52 ± 0.54 | 2.96 ± 0.65 | 1.39 ± 0.40 | |
DPIAL | 19 ± 3.78 | 18.89 ± 3.52 | 14.8 ± 3.67 | 10.94 ± 2.08 | 8.49 ± 2.2 | |
DPRAH | 2.29 ± 0.66 | 2.42 ± 0.51 | 2.52 ± 0.54 | 2.96 ± 0.65 | 1.39 ± 0.4 | |
DPRAL | 55.05 ± 9.15 a | 44.72 ± 7.90 a, b | 31.54 ± 6.25 b, c | 18.54 ± 4.30 c | 2.65 ± 0.67 c |
IP–integrity of plasma membrane, IA–integrity of acrosome, HP–high mitochondrial membrane potential (Δψm), IPIA–simultaneous integrity of plasma and acrosome membranes, IPRA–simultaneous integrity of plasma membrane e reacted acrosome, DPIA–simultaneous damage of plasma membrane and integrity of acrosome, DPRA–simultaneous damage of plasma and acrosome membranes, IPIAH–simultaneous plasma and acrosome membrane integrity and high Δψm, IPIAL—simultaneous plasma and acrosome acrosomal integrity and low Δψm, IPRAH—simultaneous plasma membrane integrity, reacted acrosome and high Δψm, IPRAL—simultaneous plasma membrane integrity, reacted acrosome and low Δψm, DPIAH—simultaneous damaged plasma membrane, acrosome integrity and high Δψm, DPIAL—simultaneous damaged plasma membrane, acrosome integrity and low Δψm, DPRAH—simultaneous damaged plasma membrane, reacted acrosome and high Δψm, DPRAL—simultaneous damaged plasma membrane, reacted acrosome and low Δψm.
1Dual stain was performed by the association of Hoechst 33342 with propidium iodide (PI for IP analysis), or
2Triple stain was performed by the association of Hoechst 33342 with PI and PSA
3Fourfold stain was performed by the association of Hoechst 33342 with PI, PSA and JC-1.
Different letters represent a significant difference (p < 0.05).
The process of centrifugation pre-freezing did not affect (p > 0.05) any sperm kinematics variables. The addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improves (p < 0.05) its TM and PM (
(A) Total and progressive motility; (B) VCL—curvilinear velocity, VSL—straight-line velocity, VAP—average path velocity; C) LIN—linearity, STR—straightness; (D) ALH—amplitude of lateral head displacement, BCF—beat cross frequency and HIPER—hypermotility. CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters represent a significant difference (p < 0.05). CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters in the same row represent a difference (p < 0.05) between treatments at the same time.
(A) Total and progressive motility; (B) VCL—curvilinear velocity, VSL—straight-line velocity, VAP—average path velocity; (C) LIN—linearity, (D) ALH—amplitude of lateral head displacement, BCF—beat cross frequency and HIPER—hypermotility. Different letters represent a significant difference (p < 0.05).
CT | CS | CW | CWSP | |
---|---|---|---|---|
5 | 83.02 ± 0.58b | 86.31 ± 1.04a | 87.04 ± 0.7a | 86.87 ± 0.91a |
60 | 82.26 ± 1.11a | 81.65 ± 1.05a | 82.56 ± 1.18a | 81.19 ± 1.08a |
120 | 34.73 ± 7.65a,b | 36.82 ± 7.63a,b | 25.58 ± 7.5b | 48.87 ± 6.37a |
Membrane integrity evaluated by sperm fourfold stain has no interaction (p > 0.05) between time vs. treatment, thus these effects were separately studied. Pre-freezing centrifugation was not deleterious (p > 0.05) for the sperm populations presenting plasma and acrosomal membrane integrity and high Δψm (IPIAH). This cell category was not improved (p > 0.05) by SP-SRF addition to the thawing medium (
Tyrosine phosphorylation on the sperm surface has no (p > 0.05) interaction between time vs. treatment, thus this effects were separately studied. Tyrosine phosphorylation was not affect (p > 0.05) by pre-freezing centrifugation process. Mean fluorescence intensity of anti-phosphotyrosine antibody conjugated to fluorescein did not decrease due to the addition of SP-SRF to FT boar semen, and it was also unchanged by any of the treatments (p > 0.05 –
CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma. Different letters represent a significant difference (p < 0.05).
Frozen-thawed boar semen fertility (CT—10.59 ± 3.92; CW—9.57 ± 4.92; CWSP—21.29 ± 7.37; p = 0.2225) was not improved by the addition of SP-SRF, and the absence of SP-SRF was not harmful to fertility (p > 0.05). Additionally, embryo recovery was similar between treatments (CT– 74.6 ± 9.76; CW– 76.08 ± 6.57; CWSP– 58.28 ± 9.09; p = 0.1864).
Cryopreservation leads to structural injuries to both sperm plasma and acrosomal membranes as well as functional damage due to capacitation-like changes, which decrease the fertilizing potential of sperm [
Furthermore, the simultaneous assessment of mitochondrial potential, plasma and acrosome membrane integrity by sperm fourfold stain is a great alternative to dual or triple stain evaluation of sperm membranes function. In general, the standard sperm analysis is performed as a dual stain in which a single sperm compartment was evaluated or triple stain that allows the evaluation of two sperm compartments (plasma and acrosome membranes) simultaneously [
Although SP withdrawal results in changes in sperm kinetics [
Semen cryopreservation drastically damages boar sperm cells [
Increased tyrosine phosphorylation appears to be a capacitation cascade event [
Seminal plasma removal is a widespread procedure in state-of-the-art boar semen cryopreservation protocols; it is essential to concentrate samples [
Some aspects of sperm physiology have been highly correlated with seminal plasma proteins. Members of the spermadhensin superfamily, the most abundant (90%) proteins in boar seminal plasma [
In conclusion, (1) the simultaneous flow cytometry assay of plasma and acrosomal membrane integrity and mitochondrial membrane potential can be achieved with Hoechst 33342, propidium iodide, PSA-FITC and JC-1 staining. (2) Addition of SP-SRF on FT boar semen improves motility characteristics of boar semen cryopreserved in absence of SP-SRF, without effect on plasma and acrossomal membrane integrity, mitochondrial potential as well as tyrosine phosphorylation. Absence of SP-SRF is not harmful to integrity of acrosomal and plasma membranes, neither for mitochondrial potential. Moreover, absence of SP-SRF did not alter tyrosine phosphorylation on sperm surface. Although SP-SRF absence was not able to reduce fertility rates, females inseminated with samples containing SP-SRF had 2.2 more fertilized structures than those inseminated with samples not containing SP-SRF. (3) Beyond that, centrifugation has not effect on the post-thawed boar semen quality, enabling separation of the centrifugation and the SP-SRF effects. Additional studies of whole sperm-rich seminal plasma addition are needed to clarify these effects.
(A) Sample subjected to three cycles of flash freezing (FF) in liquid nitrogen and then slowly thawed to induce damage to the plasma membranes followed by propidium iodide staining (PI positive). (I) Histogram showing the absence of PSA-FITC fluorescence; (II) histogram showing the absence of JC-1 orange fluorescence; (III) histogram showing the IP (plasma membrane integrity, PI negative) and DP (damaged plasma membrane, PI positive) gates and the concentration of the sperm population on the DP gate; (IV) double dot plot for evaluating the mitochondrial membrane potential (Δψm—y axis) and acrosome integrity (x axis); the green dot plot originated from the IP gate, and the red dot plot originated from the DP gate; the populations represented in the dot plots match the sperm characteristics shown in previous histograms. (B) Sample subjected to FF, as described above, to induce acrosomal damage and stained with
(TIF)
CT—control; CS—centrifuged and suspended in autologous seminal plasma (SP); CW—centrifuged and withdrawn SP; CWSP—CW containing autologous seminal plasma.
(ZIP)