Antisperm antibodies and sperm function in bulls undergoing scrotal insulation

in Reproduction
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Maria S FerrerDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Roberto PalomaresDepartment of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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David HurleyDepartment of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Anna-Claire BullingtonDepartment of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Alejandro Hoyos-JaramilloDepartment of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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João H BittarDepartment of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Correspondence should be addressed to M S Ferrer; Email: msferrer@uga.edu

(J H Bittar is now at Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA)

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Bovine antisperm antibodies (ASAs) have been associated with teratospermia and asthenospermia. It was hypothesized here that scrotal insulation induces the formation of ASAs and deterioration of sperm function. Scrotal insulation bags were placed in 10 bulls for 8 days. Semen was collected on days −29, −22 and −2, twice weekly from days 5 to 54, and thereafter weekly until day 96 (day 0 = first day of scrotal insulation). On each collection day, scrotal circumference, sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, mitochondrial membrane potential, ASA binding and DNA integrity were evaluated. The percentage of IgG- and IgA-bound sperm increased between days 12 and 96 (P < 0.0001), in association with poor motility (days 19–30, P < 0.005) and morphology (days 8–40, P < 0.0001). Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001). There was also a deterioration in sperm membrane integrity (days 19–40, P < 0.0001), acrosome integrity (days 26–89, P < 0.0001), lipid peroxidation (days 5–12, P < 0.0001), and mitochondrial membrane potential (days 12–96, P = 0.001). In contrast, a decrease in apoptotic cells (days 37–83, P = 0.0002) and lipid peroxidation (days 19–96, P < 0.0001) was noticed. Most bulls recovered normospermia by day 96. However, the persistence of ASAs, acrosomal damage and dysfunctional mitochondria suggest a long term effect of scrotal insulation on sperm function and the homeostasis of the reproductive immune system.

Abstract

Bovine antisperm antibodies (ASAs) have been associated with teratospermia and asthenospermia. It was hypothesized here that scrotal insulation induces the formation of ASAs and deterioration of sperm function. Scrotal insulation bags were placed in 10 bulls for 8 days. Semen was collected on days −29, −22 and −2, twice weekly from days 5 to 54, and thereafter weekly until day 96 (day 0 = first day of scrotal insulation). On each collection day, scrotal circumference, sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, mitochondrial membrane potential, ASA binding and DNA integrity were evaluated. The percentage of IgG- and IgA-bound sperm increased between days 12 and 96 (P < 0.0001), in association with poor motility (days 19–30, P < 0.005) and morphology (days 8–40, P < 0.0001). Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001). There was also a deterioration in sperm membrane integrity (days 19–40, P < 0.0001), acrosome integrity (days 26–89, P < 0.0001), lipid peroxidation (days 5–12, P < 0.0001), and mitochondrial membrane potential (days 12–96, P = 0.001). In contrast, a decrease in apoptotic cells (days 37–83, P = 0.0002) and lipid peroxidation (days 19–96, P < 0.0001) was noticed. Most bulls recovered normospermia by day 96. However, the persistence of ASAs, acrosomal damage and dysfunctional mitochondria suggest a long term effect of scrotal insulation on sperm function and the homeostasis of the reproductive immune system.

Introduction

The antigenicity of spermatozoa was first suspected in 1899, when sperm immobilizing factors were identified in guinea pigs immunized with bull sperm (Landsteiner 1899). Since then, several authors have confirmed the ability of bull spermatozoa to induce an autoimmune response, epididimo-orchitis and antisperm antibodies (ASAs) (Menge et al. 1962, Hunter & Hafs 1964, Losos et al. 1968, Menge & Christian 1971, Parsonson et al. 1971, Wright et al. 1980, Kim et al. 1999). Presence of ASAs in serum or seminal plasma was described in bulls with seminal vesiculitis (Perez & Cuellar Carrasco 1964), chronic orchitis (Vlok et al. 2009) and genital infection with Chlamydophila sp. (Zralý et al. 1998), Brucella abortus and bovine herpes virus (Hegazi & Ezzo 1995). Serum ASAs have also been associated with reduced pregnancy rates (Zralý et al. 2002, Kuntareddi et al. 2020) and in vitro fertilization rates in cattle (Kim et al. 1999).

Recently, the presence of ASAs bound to the sperm membrane at ejaculation (sperm-bound ASAs) was described in bulls (Ferrer et al. 2015). Non-satisfactory breeder bulls had a higher prevalence of sperm-bound IgA than satisfactory breeders (Ferrer et al. 2015). In addition, the presence of sperm-bound IgA and IgG was associated with teratospermia and asthenospermia (Ferrer et al. 2015). It can be speculated that the testicular dysfunction was caused by a primary autoimmune orchitis, leading to abnormal semen quality and ASAs. On the other hand, testicular trauma, infection, neoplasia or heat stress could result in disruption of the testicular homeostasis and the immune-privileged status, resulting in secondary autoimmune orchitis and ASAs.

The most common cause of declining semen quality in bulls is testicular degeneration (Barth 2015). The most studied cause of testicular degeneration is thermal injury, which can result from increased environmental temperature, fever, local inflammation, or impaired thermoregulation due to abnormalities of the spermatic cord, scrotal skin or vaginal cavity. An increase in testicular temperature leads to Sertoli and Leydig cell dysfunction, and loss of the germinal epithelium (Kastelic 2015). Testicular heat stress results in an imbalance in antioxidant/oxidant homeostasis, leading to increased levels of reactive oxygen species (ROS) and oxidative stress, which induces germ cell apoptosis and decreases germ cell proliferation (Paul et al. 2009, Kanter et al. 2013, Duramayanthi et al. 2015). The decline in sperm morphology and motility associated with heat-induced testicular degeneration has been described in the bull (Vogler et al. 1991, Barth & Bowman 1994, Brito et al. 2003, Arteaga et al. 2005, Rahman et al. 2011). However, the mechanisms leading to those changes are poorly understood, and the contribution of the immune system to testicular dysfunction has not been evaluated.

It was hypothesized, in this study, that heat-induced testicular degeneration results in the formation of ASAs in association with deterioration of sperm functional and structural parameters. The objective was to evaluate the presence and changes in ASAs, and sperm function and morphology associated with scrotal insulation.

Materials and methods

Animals and experimental design

The study was performed under the approval and supervision of the University of Georgia’s Institutional Animal Care and Use Committee. The animals were housed together in the pasture at the University of Georgia Double Bridges Farm, Department of Animal Sciences, in Athens-Clarke County, GA, USA. Water and Bermuda grass hay were fed ad libitum. Ten 15- to 18-month-old SimAngus bulls were purchased from local producers. At the time of purchase, all bulls were classified as satisfactory potential breeders based on guidelines from the Society for Theriogenology (see ‘Breeding soundness examination, semen collection and semen evaluation’ section; Koziol & Armstrong 2018).

After 1 week of acclimation, three ejaculates were collected from each bull 29, 22 and 2 days prior to placing the scrotal insulation bags (day 0 = first day of scrotal insulation) to establish baseline semen parameters. The scrotal insulation bags were custom-made out of sleeping bags. They had an inner layer of polycotton, a middle layer of synthetic insulation material, and an outer layer of waterproof nylon. A roll of gauze was threaded around the neck of the bag, and was used to keep the bags in place by tying them around the neck of the scrotum. The tightness of the lace was adjusted after 10 min of initial placement. The bulls were evaluated visually twice a day to ensure the bags were in place. The scrotal insulation bags were removed on day 8. Semen was collected with electroejaculation twice a week from days 5 to 54, and thereafter weekly until day 96. At the time of each semen collection, except for day 5 when insulation bags were in place, scrotal circumference was measured. A 0.5 mL aliquot of semen was immediately frozen on dry ice, and stored at −70°C, for evaluation of DNA integrity. Sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, antisperm antibody binding, mitochondrial membrane potential and DNA integrity were evaluated (see ‘Breeding soundness examination, semen collection and semen evaluation’ section). Pre-insulation values served as each bull’s own control. At the end of the study, bulls were castrated and sold at the local sale barn as non-breeding animals.

Breeding soundness examination, semen collection and basic semen evaluation

Scrotal circumference was determined with a scrotal measuring tape. The scrotal contents, prepuce and penis were evaluated by visual inspection and palpation. The accessory sex glands were palpated per rectum. The sperm-rich fraction of semen was collected using the programmed settings of an electroejaculator (Pulsator IV, Lane Manufacturing Inc., Denver, CO, USA). An aliquot of semen was diluted in warm Dulbecco’s phosphate buffer solution (DPBS) to a concentration of 50 x 106 spermatozoa/mL for assessment of sperm motility with a computer-assisted semen analyzer (CASA; SpermVision Professional, Minitube of America, Verona, WI, USA). The settings of the instrument were: field depth of view 20 μm, pixel to μm ratio 130–100, cell area 18–80 μm, frames acquired 30, frame rate 60 Hz, AOC cut off static cells 5 and DSL cut off 4.5 µm/s (progressive motility). Semen was placed in a 20-µL chamber over the heated stage at 38°C. Mean percentage of total and progressively motile spermatozoa was assessed from all cells present in seven fields with a 20x phase-contrast objective. Other motion parameters reported were average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), curvilinear distance (DCL), straight-line distance (DSL), and distance of average path (DAP).

For the assessment of sperm morphology, spermatozoa were diluted 1:10 (v:v) with formalin buffered solution, and a 5-μL drop was placed onto a microscope slide. Evaluation was done with phase-contrast microscopy at 100x magnification under oil immersion, and 100 spermatozoa were classified based on their morphological characteristics. Abnormalities included those of the acrosome, head, midpiece, proximal droplets, coiled tails, and detached heads (Koziol & Armstrong 2018).

Bulls were classified as satisfactory breeders if they met the following requirements (Koziol & Armstrong 2018): no gross genital pathology, ≥30% individual sperm motility, ≥70% morphologically normal spermatozoa and minimum scrotal circumference of 31 cm. Bulls not meeting at least one of these criteria were classified as non-satisfactory breeders.

Staining for evaluation of sperm functional parameters

For analysis of ASA binding, 2.5 x 106 of washed spermatozoa were added to each of four tubes containing 320 µL of Dulbecco’s phosphate buffer solution (DPBS). Antibodies were added to each tube: IgG = 30 µL of FITC-labeled polyclonal goat anti-bovine IgG F(ab’)2 (12.5 µg/mL; Cat. No. 101-096-003, Jackson Immunoresearch Laboratories Inc.); IgG negative control = 30 µL of FITC-labeled polyclonal rabbit anti-goat IgG F(ab’)2 (12.5 µg/mL; Cat. No. 305-096-003; Jackson Immunoresearch Laboratories Inc.); IgA = 20 µL of FITC-labeled polyclonal rabbit anti-bovine IgA (12.5 µg/mL; Cat. No. A10-108F; Bethyl Laboratories, Montgomery, TX, USA); or IgA negative control = 20 µL of FITC-labeled polyclonal goat anti-mouse IgA (12.5 µg/mL; Cat. No. A90-103F; Bethyl Laboratories). The samples were incubated for 30 min at room temperature, followed by one wash by centrifugation. Propidium iodide (PI), 3 µL of a 1.2 mM solution, was added for the simultaneous staining of dead cells. The percentage of membrane-intact and membrane-damaged IgG- and IgA-bound spermatozoa was assessed with flow cytometry. Presence of ASAs in seminal plasma or serum was not evaluated.

Plasma membrane integrity was evaluated with the stains SYBR14 and PI (LIVE/DEAD kit, cat. No. L7011, Molecular Probes). First, 25 x 106 sperm were diluted to 1 mL in DPBS. Then, 5 µL of a 20 μM solution of SYBR14, and 4 µL of a 2.4 mM solution of PI were added. The samples were incubated for 10 min at 38°C, after which they were evaluated using fluorescence microscopy under a 40x objective. All spermatozoa present in 10 fields were classified as membrane-intact (green fluorescence) or membrane-damaged (red fluorescence) by a built-in software within the CASA system (SpermVision Professional, Minitube of America).

Mitochondrial membrane potential was evaluated using the fluorescent probe 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1, Cat. No. T3168, Molecular probes). One microliter of JC-1 stock solution was added to 1 ml of sperm suspension containing 5 x 106 spermatozoa, together with 8 μL of a 1.2 mM solution of PI. Samples were incubated at 38°C for 15 min, and were evaluated using fluorescence microscopy under a 40x objective. One hundred membrane-intact cells were classified as having high (orange-yellow) or low (green) mitochondrial membrane potential.

Acrosome integrity was evaluated with fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA; Cat. No. F-2301-1, EY Laboratories, San Mateo, CA, USA) and PI. One microliter of a 1 µg/mL solution of FITC-PNA and 1 µL of a 6 μM solution of propidium iodide (PI; Live/Dead Kit, Molecular Probes) were added to a 1-mL suspension containing 5 x 106 spermatozoa. Semen was incubated at 38°C for 10 min. The percentage of acrosome-damaged membrane-intact spermatozoa was evaluated with flow cytometry.

Sperm apoptosis was evaluated using FITC-labelled Annexin V stain (ANV, Cat. No. A13199, Molecular Probes). Spermatozoa were washed by centrifugation at 600 g for 5 min and were resuspended to 4 x 105/mL in binding buffer (0.1 M HEPES, 1.4 M sodium chloride, 25 mM calcium chloride in distilled water, pH 7.4). Then, 10 µL of FITC-Annexin V was added to 1 mL of sperm suspension, and semen was incubated for 15 min at room temperature. Semen was then washed by centrifugation to remove unbound stain, and was resuspended to 0.5 mL in binding buffer. Then, 0.5 µL of a 6 μM solution of PI was added. The percentage of membrane-intact apoptotic cells was evaluated with flow cytometry.

Susceptibility to sperm membrane lipid peroxidation was evaluated with the fluorescent probe BODIPY C11 (cat. No. M24571, Molecular Probes). Semen was washed by centrifugation once. Then, 0.5 μL of a 2 mM solution of BODIPY C11 was added to 1 mL of suspension containing 5 x 106 spermatozoa. The sample was incubated at 38°C for 30 min. After washing by centrifugation to remove unbound dye, the percentage of cells with lipid peroxidation was evaluated with flow cytometry.

Sperm DNA integrity was evaluated using a sperm chromatin structure assay. Snap-frozen semen was thawed in a water bath at 35°C for 30 s. Acid-detergent solution (2.19 g NaCl, 1 mL 2N HCl solution, 0.25 mL Triton X-100, q.s. 250 mL deionized water, pH 1.2), 400 μL, was added to a suspension of 0.5 x 106 sperm in 200 μL of TNE buffer solution (0.186 g disodium ETDA, 0.79 g Tris–HCL, 4.380 g NaCl in 500 mL deionized water, pH 7.4) After 30 s, 1.2 mL of a 2 μg/mL acridine orange (Molecular Probes, Cat. No. A3568) working solution (diluted in buffer: 3.8869 g citric acid monohydrate, 8.9429 g Na2HPO4, 4.3850 g NaCl, 0.1700 g disodium EDTA, q.s. 500 mL water, pH 6) was added. The DNA fragmentation index (DFI) was calculated as the percentage of cells with fragmented DNA (red fluorescence) over total (red + green) fluorescent cells using flow cytometry (Evenson 2016).

Flow cytometry

Fluorescence-activated flow cytometry (Accuri C6 Plus, BD Biosciences) was used for the evaluation of most sperm functional parameters. From each sample, 10,000 events in the forward and 90º light scatter population representing whole sperm were analyzed using 0.2-μm filtered 18 mega-ohm ultra-pure water as the sheath fluid. A gate containing spermatozoa was selected based on dot plot distribution of forward (size) vs side scatter (complexity parameter) to eliminate debris and somatic cells from the analysis. The green and red signals were detected using a 5 mW blue argon laser (488 nm) and emission filters (535 ± 30 nm for green and 585 ± 30 nm for red). Baseline background fluorescence signal was initially evaluated in unstained samples. The control area was marked on unstained samples with <1% of cells registering as positive for both signals. Compensation for green emission into the red or orange detector or vice versa was done by establishing quadrants on spermatozoa labeled with individual fluorochromes, followed by electronic subtraction of the green emission into the red or orange detector, and red or orange emission into the green detector. After color compensation, fluorescence emission data were collected with logarithmic amplification for green fluorescence (FITC, BODIPY and acridine orange using FL1 detector), orange fluorescence (BODIPY and acridine orange using FL2 detector), and red fluorescence (PI using FL3 detector).

Statistical analysis

Statistical analysis was performed using the software Statistical Analysis System (SAS® Institute, Cary, NC, USA). Distribution of the data was tested for normality using the Shapiro–Wilk test. Variables that did not follow a normal distribution underwent logarithmic transformation. The effect of time was evaluated using a mixed-effect model, with a fixed effect of time and a random effect of bull. Where an effect of time was detected, multiple comparisons were performed using leastsquare means and a Bonferroni adjustment. While all pairwise comparisons were performed and included in the model, differences with pre-insulation values were reported to focus on the time of appearance of changes and return to baseline values. Each bull was used as its own control by comparing changes to pre-insulation values. Values were then back transformed and expressed as geometric means and s.e.m. The frequency of satisfactory breeder bulls was compared among times using a Chi square test. Differences were considered significant if P < 0.05.

Results

Scrotal insulation induced an antisperm antibody response in 9/10 bulls. The mean percentage of IgG- and IgA-bound sperm increased on day 12, and remained higher than pre-insulation values until the end of the study on day 96 (P < 0.0001) (Fig. 1A and B). An early, transient increase was observed in the percentage of membrane-intact IgA-bound sperm on day 5 (Fig. 1A). Increased binding of IgG and IgA occurred to both membrane-intact and membrane-damaged sperm. However, IgA binding was more pronounced in membrane-damaged sperm (Fig. 1A). Examples of dot plot and histogram distributions of spermatozoa labeled with anti-goat and anti-bovine IgA, and corresponding to one bull before and after scrotal insulation are shown in Fig. 2.

Figure 1
Figure 1

Percentage of spermatozoa with the binding of IgA (panel A) or IgG (panel B) before and after scrotal insulation for 8 days (days 0–8) (mean ± s.e.m.). Symbols indicate a difference with pre-insulation values in the percentage of ASA-bound membrane-damaged (^) and membrane-intact (*) spermatozoa (P < 0.0001).

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

Figure 2
Figure 2

Dot plot distribution of spermatozoa labeled with anti-goat IgA (panel A, negative control) and anti-bovine IgA (panel B) prior to scrotal insulation. Panel C represents a histogram with anti-bovine IgA labeled spermatozoa gated on the membrane-intact population (PI negative). The bottom panels show the dot plot distributions of spermatozoa labeled with anti-goat IgA (panel D, negative control) and anti-bovine IgA (Panel E) 89 days after scrotal insulation in the same bull. Panels F and G represent histograms with anti-bovine IgA labeled spermatozoa gated on the membrane-intact and membrane-damaged populations, respectively.

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

The increase in ASA binding was associated with changes in semen quality. Total (P = 0.0001) and progressive (P = 0.0002) sperm motility declined between days 19 and 30 (Fig. 3A), together with sperm distance (DCL, P = 0.012; DAP, P = 0.006; DSL, P = 0.022; Fig. 3B) and velocity (VCL, P = 0.007; VAP, P = 0.003; VSL, P = 0.011; Fig. 3C) parameters.

Figure 3
Figure 3

Sperm motility (panel A), distance (panel B) and velocity parameters (panel C) before and after scrotal insulation for 8 days (days 0–8). DCL, mean curvilinear distance; DAP, distance of average path; DSL, straight line distance; VCL, curvilinear velocity; VAP, average path velocity; VSL, straight-line velocity. Symbols indicate a significant difference with pre-insulation values (P < 0.05; mean ± s.e.m.).

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

The percentage of morphologically normal sperm decreased compared with pre-insulation values between days 8 and 40 (P < 0.0001) (Fig. 4A). This was associated with increases in sperm head abnormalities (days 15–89, P = <0.0001; Fig. 4B), acrosome abnormalities (days 12–37, P = 0.006; Fig. 4C), abnormal midpieces (days 15–44, P < 0.0001; Fig. 4C), proximal droplets (days 30–44, P = 0.009; Fig. 4D), detached heads (days 5–96, P < 0.0001; Fig. 4D), and coiled tails (days 15–96, P < 0.0001; Fig. 4D).

Figure 4
Figure 4

Percentage of morphologically normal sperm (panel A), and sperm morphological abnormalities (panels B–D) before and after scrotal insulation for 8 days (days 0–8). Symbols indicate a difference from pre-insulation values (P < 0.05; mean ± s.e.m.).

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001; Fig. 5A). Thereafter, scrotal circumference increased steadily to exceed pre-insulation values on the last day of the study. In spite of this decrease in mean values, scrotal circumference remained above minimum recommended values for satisfactory breeders in all bulls at all times (31 cm).

Figure 5
Figure 5

Scrotal circumference (panel A), and a number of bulls classified as satisfactory breeders (panel B) before and after scrotal insulation for 8 days (days 0–8). *Means differed from pre-insulation values (P < 0.05; mean ± s.e.m.).

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

The changes in semen quality resulted in changes in the frequency of bulls classified as satisfactory breeders with time (P < 0.0001). The lowest frequency occurred between days 15 and 33. On day 68 (one spermatogenic cycle after scrotal insulation), 5/10 bulls had recovered the satisfactory breeder classification. At the end of the study, two bulls were still considered non-satisfactory breeders (Fig. 5B).

The percentage of sperm with intact membranes decreased between days 19 and 40 (P = 0.001; Fig. 6A). The percentage of sperm with high mitochondrial membrane potential also decreased on day 12 until the end of the study (P = 0.001; Fig. 6B). The percentage of membrane-intact sperm with damaged acrosomes increased intermittently between days 26 and 89 (P < 0.0001; Fig. 6C). The percentage of sperm with lipid peroxidation initially increased on days 5 and 12, but subsequently decreased from day 19 until the end of the study (P < 0.0001; Fig. 6D). A decrease in the percentage of membrane-intact sperm with apoptotic changes (i.e. phosphatidylserine externalization) was also seen on days 37, 61 and 82 (P = 0.0002; Fig. 6E). The DNA fragmentation index increased between days 15 and 40 (P < 0.0001; Fig. 7A,B and C).

Figure 6
Figure 6

Percentage of sperm with the intact membrane (panel A), high mitochondrial membrane potential (HMMP, panel B), damaged acrosome (panel C), lipid peroxidation (panel D) and apoptosis (panel E) before and after scrotal insulation for 8 days (days 0–8). *Means differed from pre-insulation values (P < 0.05; mean ± s.e.m.).

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

Figure 7
Figure 7

The DNA fragmentation index (DFI) increased between days 15 and 40 after scrotal insulation for 8 days (days 0–8) (P < 0.0001; mean ± s.e.m.; panel A). *Indicates significant difference compared with pre-insulation values. The scattergram in panel B corresponds to a pre-insulation sample (low DFI), while panel C corresponds to the same bull 26 days after scrotal insulation (high DFI). Cells with fragmented DNA are contained within the population in H2-3.

Citation: Reproduction 160, 5; 10.1530/REP-20-0207

Discussion

This is the first study to demonstrate that heat-induced testicular degeneration results in an increased percentage of IgG- and IgA-bound spermatozoa in bulls. This increase was first evident 12 days after scrotal insulation, and persisted for 96 days, when the study ended. The timing of appearance of sperm-bound ASAs after scrotal insulation was similar to that described after isoimmunization with sperm antigens (Menge & Christian 1971, Parsonson et al. 1971, Wright 1980). Infiltration of the rete testis, efferent ducts, epididymis and ampulla with neutrophils, lymphocytes and macrophages was first observed 10 days after immunization (Menge & Christian 1971, Parsonson et al. 1971). The rete testis, efferent ducts and ampulla were proposed as the locations where bovine ASAs leak from the blood into the genital tract, or where local production occurs due to lack of a blood-testis barrier (Parsonson et al. 1971). Given the duration of scrotal insulation here, spermatozoa ejaculated before day 15 were in the epididymis or ampulla at the time of injury (Barth & Bowman 1994, Brito et al. 2003), where initial ASA binding may have occurred. Sperm ejaculated between days 15 and 22 represent an overlap of cells that were injured at the testes and the epididymides during scrotal heating, while sperm defects arising thereafter resulted from testicular changes (Barth & Bowman 1994). Binding of ASAs after 15 days may have occurred at the testes or the excurrent duct system.

The reason for the higher binding of IgA to cells with damaged membranes may have physiological implications, since ASAs may act as opsonins or markers, targeting damaged cells for removal from the male or female reproductive tract. In reproductively normal bulls, there is a selective removal of defective sperm during passage through the excurrent duct system, with a progressive decrease in the number of abnormal sperm between the efferent ducts and the ampulla (Ramamohana et al. 1980). Removal is most likely accomplished by phagocytosis by epithelial cells and resident macrophages (Sinowatz et al. 1979). In bulls with experimentally-induced ASAs, macrophages within the ampulla often contained phagocytosed sperm (Parsonson et al. 1971). However, the efficiency of removal is affected by the number of abnormal sperm entering the excurrent ducts. Bulls with severe sperm abnormalities failed to efficiently remove abnormal sperm, resulting in teratospermia (Ramamohana et al. 1980). Increased binding of IgA and IgG to membrane-intact sperm was also detected. The median percentage of IgA- and IgG-bound membrane-intact sperm among ASA-positive samples was 22.3% (range 12.7–52.2%) and 25.1% (range 20.2–66.4%), respectively. The cutoff percentage of ASA-bound sperm at which point a negative effect occurs on bull fertility is not known. It is assumed that at least 70% of competent spermatozoa are needed to ensure the formation of an adequate sperm reservoir (Flowers 2013). The values observed in this study can reduce the number of competent sperm in an insemination dose, reducing fertility.

Eight bulls, in this study, recovered normospermia and the satisfactory breeder classification by day 96. However, the presence of sperm-bound IgG and IgA still persisted. Similarly, in immunized bulls, the inflammatory changes and serum ASAs persisted for up to 146 days (Parsonson et al. 1971, Wright 1980). In chronic cases, there was obstruction of the rete testis or tubules in the head of the epididymis with aggregates of leucocytes, sperm and round germ cells, causing rupture of the epididymal tubules and sperm granulomas (Menge & Christian 1971, Parsonson et al. 1971). Sperm granulomas were associated with chronic inflammation and the persistence of ASAs in the face of normospermia (Parsonson et al. 1971). The persistence of ASAs, especially those bound to membrane-intact sperm, in normospermic bulls after recovery from testicular degeneration has clinical and economic implications. These bulls would have been considered appropriate to enter a breeding program based on a routine breeding soundness examination. However, the presence of bovine ASAs has been associated with decreased conception rate (Zralý et al. 2002, Kuntareddi et al. 2020), decreased ability of spermatozoa to bind to oviductal epithelial cells (Ferrer et al. 2016), and reduced ability to undergo capacitation and bind to the zona pellucida (Ferrer et al. 2017). Therefore, bulls that recover from transient testicular degeneration may still have poor fertility in spite of a positive classification during routine breeding soundness examination. One of the limitations of this study is the lack of assessment of fertility, which deserves further evaluation.

The pathophysiology of ASA formation is not known in the bull. In rodents, dysregulation of the tolerogenic testicular environment leads to the development of autoimmune orchitis and the formation of ASAs. This is initiated by the release of damage-associated molecular patterns (DAMPs) by damaged germ cells, which induce pro-inflammatory cytokine production by Sertoli cells through Toll-like receptor (TLR) activation (Fijak et al. 2005, Hedger 2011, Liu et al. 2015). Furthermore, activation of TLRs in germ cells directly induces apoptosis (Hedger 2011). It is possible to speculate that testicular heat stress may have caused the release of ROS, leading to potential germ cell apoptosis and release of DAMPs, as reported previously (Paul et al. 2009, Duramayanthi et al. 2015, Kaur & Bansal 2015). Another limitation of this study is that the production of ROS was not evaluated, and the interpretation of oxidative damage is limited since BODIPY C11 only detects lipid peroxidation occurring after incorporation of the probe into the sperm membrane. The higher susceptibility of sperm to lipid peroxidation on days 5 and 12 could be due to aberrant production of ROS by, or a deficient antioxidant system of, defective sperm suffering the effect of heat stress within the epididymides (Aitken & Clarkson 1987, Aitken et al. 2007). On the other hand, the decrease in susceptibility to lipid peroxidation after day 19 is similar to previous reports (Losano et al. 2018). Environmental heat stress results in a decrease in polyunsaturated fatty acids and increase in cholesterol content in the sperm membrane (Argov et al. 2007, Argov-Argaman et al. 2013). While the temperature was not measured in this study, previous work demonstrated that scrotal insulation elevates scrotal temperature above 35°C (normal range 33–34.5°C) (Wildeus & Entwistle 1983, Barth & Bowman 1994). It is possible that insulation–induced heat stress also led to changes in membrane lipid composition that decreased the susceptibility to lipid peroxidation. Membrane lipid composition was not assessed here and deserves further evaluation.

Following scrotal insulation, there was a transient deterioration in sperm morphology (days 12–40), motility (days 19–30) and membrane integrity (days 12–40). The timing of appearance of these alterations was similar to previous reports (Vogler et al. 1991, Barth & Bowman 1994, Brito et al. 2003, Arteaga et al. 2005, Rahman et al. 2011), and likely resulted from damage to spermatids and meiotic cells (Barth & Bowman 1994, Brito et al. 2003). This deterioration resulted in the bulls being classified as non-satisfactory potential breeders. Even though the percentage of morphologically normal sperm was not statistically different from pre-insulation values after day 44, it must be noted that this parameter was still below minimum recommended values for satisfactory breeder bulls (Koziol & Armstrong 2018). It is generally recommended to retest bulls 60 days after the condition causing testicular dysfunction has resolved. This allows for one spermatogenic cycle to occur, with the idea that unaffected sperm would be ejaculated after restoration of testicular function (Koziol & Armstrong 2018). However, only 5/10 of the bulls in this study had recovered normospermia 60 days after the end of scrotal insulation. Moreover, two bulls still had not recovered normospermia on day 96 (1½ spermatogenic cycles). This was due to persistently increased percentages of sperm with abnormal heads, detached heads and coiled tails. Therefore, a more extended recovery period may be needed before retesting bulls to prevent unnecessary culling.

Further support of a prolonged testicular dysfunction and impaired sperm function was provided by the persistently increased percentage of sperm with damaged acrosomes, and the decreased percentage of sperm with high mitochondrial membrane potential. A similar prolonged effect of heat stress on these two parameters was described in bulls (Losano et al. 2018). The prolonged mitochondrial dysfunction in motile sperm was striking. Mitochondria play a key role in supporting sperm motility by producing ATP via oxidative phosphorylation (Freitas et al. 2017). However, bull spermatozoa are able to maintain ATP production and motility via the glycolytic pathway when their mitochondria are uncoupled (Losano et al. 2017). The glycolytic pathway takes place along the flagellum and does not depend on mitochondria (Freitas et al. 2017). Furthermore, mammalian sperm can alternate metabolic pathways depending on oxygen and substrate availability in the medium (Freitas et al. 2017). It is possible that the presence of glycolytic substrates in bull seminal plasma was able to support ATP production via glycolysis, maintaining appropriate sperm motility in spite of the loss of mitochondrial function.

The susceptibility of sperm DNA to acid denaturation increased between days 12 and 40 after scrotal insulation, as previously reported (Karabinus et al. 1997, Fernandes et al. 2008, Rahman et al. 2011, Losano et al. 2018, Boe-Hansen et al. 2020). Decreased mitochondrial membrane potential, alterations in DNA integrity and externalization of phosphatidylserine are features of sperm apoptosis (Aitken & Baker 2013). In spite of two apoptotic features being present in this study (i.e. decreased mitochondrial function and DNA integrity), the percentage of cells with externalized phosphatidylserine actually decreased after scrotal insulation. The reason for this finding is not known.

In summary, scrotal insulation induced deterioration of all sperm parameters. Concurrently with changes in semen quality, there was an increase in ASA binding. The increase in ASA-bound sperm that persisted beyond regaining normospermia likely indicates prolonged dysregulation of tolerogenic mechanisms after apparent recovery from testicular degeneration. The persistence of ASAs, acrosomal damage and dysfunctional mitochondria reflect a long term effect of scrotal insulation on testicular function, sperm function and the homeostasis of the reproductive immune system that is not detected with routine semen evaluation. These changes stress the importance of pursuing advanced diagnostic tests during the evaluation of bull breeding soundness potential, especially in sires with a history of testicular degeneration.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

Supported by financial donations from Multimin, Inc.

Author contribution statement

M S F and R P designed the study, collected and analyzed data, and wrote the manuscript. D H helped standardize assays, and collect and interpret flow cytometry data. A-C B helped with animal handling, semen processing and laboratory set up. A H-J and J H B helped with animal handling and sample collection. All authors contributed to data interpretation and manuscript writing.

Acknowledgement

The authors thank personnel from Double Bridges Farm for the support with animal housing and handling.

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    Figure 1

    Percentage of spermatozoa with the binding of IgA (panel A) or IgG (panel B) before and after scrotal insulation for 8 days (days 0–8) (mean ± s.e.m.). Symbols indicate a difference with pre-insulation values in the percentage of ASA-bound membrane-damaged (^) and membrane-intact (*) spermatozoa (P < 0.0001).

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    Figure 2

    Dot plot distribution of spermatozoa labeled with anti-goat IgA (panel A, negative control) and anti-bovine IgA (panel B) prior to scrotal insulation. Panel C represents a histogram with anti-bovine IgA labeled spermatozoa gated on the membrane-intact population (PI negative). The bottom panels show the dot plot distributions of spermatozoa labeled with anti-goat IgA (panel D, negative control) and anti-bovine IgA (Panel E) 89 days after scrotal insulation in the same bull. Panels F and G represent histograms with anti-bovine IgA labeled spermatozoa gated on the membrane-intact and membrane-damaged populations, respectively.

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    Figure 3

    Sperm motility (panel A), distance (panel B) and velocity parameters (panel C) before and after scrotal insulation for 8 days (days 0–8). DCL, mean curvilinear distance; DAP, distance of average path; DSL, straight line distance; VCL, curvilinear velocity; VAP, average path velocity; VSL, straight-line velocity. Symbols indicate a significant difference with pre-insulation values (P < 0.05; mean ± s.e.m.).

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    Figure 4

    Percentage of morphologically normal sperm (panel A), and sperm morphological abnormalities (panels B–D) before and after scrotal insulation for 8 days (days 0–8). Symbols indicate a difference from pre-insulation values (P < 0.05; mean ± s.e.m.).

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    Figure 5

    Scrotal circumference (panel A), and a number of bulls classified as satisfactory breeders (panel B) before and after scrotal insulation for 8 days (days 0–8). *Means differed from pre-insulation values (P < 0.05; mean ± s.e.m.).

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    Figure 6

    Percentage of sperm with the intact membrane (panel A), high mitochondrial membrane potential (HMMP, panel B), damaged acrosome (panel C), lipid peroxidation (panel D) and apoptosis (panel E) before and after scrotal insulation for 8 days (days 0–8). *Means differed from pre-insulation values (P < 0.05; mean ± s.e.m.).

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    Figure 7

    The DNA fragmentation index (DFI) increased between days 15 and 40 after scrotal insulation for 8 days (days 0–8) (P < 0.0001; mean ± s.e.m.; panel A). *Indicates significant difference compared with pre-insulation values. The scattergram in panel B corresponds to a pre-insulation sample (low DFI), while panel C corresponds to the same bull 26 days after scrotal insulation (high DFI). Cells with fragmented DNA are contained within the population in H2-3.

  • Aitken RJ & Baker MA 2013 Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int ernational J ournal of Dev elopmental Biol ogy 57 265272. (https://doi.org/10.1387/ijdb.130146ja)

    • Search Google Scholar
    • Export Citation
  • Aitken RJ & Clarkson JS 1987 Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J ournal of Reprod uction and Fert ility 81 459469. (https://doi.org/10.1530/jrf.0.0810459)

    • Search Google Scholar
    • Export Citation
  • Aitken RJ, Wingate JK, De Iuliis GN & McLaughlin EA 2007 Analysis of lipid peroxidation in human spermatozoa using BODIPY C11. Mol ecular Human Reprod uction 13 203211. (https://doi.org/10.1093/molehr/gal119)

    • Search Google Scholar
    • Export Citation
  • Argov N, Sklan D, Zeron Y & Roth Z 2007 Association between seasonal changes in fatty-acid composition, expression of VLDL receptor and bovine sperm quality. Theriogenology 67 878885. (https://doi.org/10.1016/j.theriogenology.2006.10.018)

    • Search Google Scholar
    • Export Citation
  • Argov-Argaman N, Mahgrefthe K, Zeron Y & Roth Z 2013 Season-induced variation in lipid composition is associated with semen quality in Holstein bulls. Reproduction 145 479489. (https://doi.org/10.1530/REP-12-0498)

    • Search Google Scholar
    • Export Citation
  • Arteaga AA, Barth AD & Brito LFC 2005 Relationship between semen quality and pixel–intensity of testicular ultrasonograms after scrotal insulation in beef bulls. Theriogenology 64 408415. (https://doi.org/10.1016/j.theriogenology.2004.12.008)

    • Search Google Scholar
    • Export Citation
  • Barth A 2015 Testicular degeneration. In Bovine Reproduction, pp. 104108. Ed Hopper. Ames, Iowa: Wiley Blackwell.

  • Barth AD & Bowman PA 1994 The sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls. Can adian Vet erinary J ournal 35 93102.

    • Search Google Scholar
    • Export Citation
  • Boe‐Hansen GB, Rêgo JPA, Satake N, Venus B, Sadowski P, Nouwens A, Li Y & McGowan M 2020 Effects of increased scrotal temperature on semen quality and seminal plasma proteins in Brahman bulls. Mol ecular Reprod uction and Dev elopment 87 574597. (https://doi.org/10.1002/mrd.23328)

    • Search Google Scholar
    • Export Citation
  • Brito LFC, Silva AEDF, Barbosa RT, Unanian MM & Kastelic JP 2003 Effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus × Bos taurus bulls. Anim al Reprod uction Sci ence 79 115. (https://doi.org/10.1016/s0378-4320(03)00082-4)

    • Search Google Scholar
    • Export Citation
  • Duramayanthi D, Agarwal A & Ong C 2015 Causes, effects and molecular mechanisms of testicular heat stress. Reprod uctive Biomed icine Online 30 1427. (https://doi.org/10.1016/j.rbmo.2014.09.018)

    • Search Google Scholar
    • Export Citation
  • Evenson DP 2016 The Sperm chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Anim al Reprod uction Sci ence 169 5675. (https://doi.org/10.1016/j.anireprosci.2016.01.017)

    • Search Google Scholar
    • Export Citation
  • Fernandes CE, Dode MAN, Pereira D & Silva AEDF 2008 Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability. Theriogenology 70 15601568. (https://doi.org/10.1016/j.theriogenology.2008.07.005)

    • Search Google Scholar
    • Export Citation
  • Ferrer MS, Laflin S, Anderson DE, Miesner MD, Wilkerson MJ, George A, Miller LMJ, Larson R & Garcia Flores EO 2015 Prevalence of bovine sperm-bound antisperm antibodies and their association with semen quality. Theriogenology 84 94100. (https://doi.org/10.1016/j.theriogenology.2015.02.017)

    • Search Google Scholar
    • Export Citation
  • Ferrer MS, Anderson DE, Miller LMJ, George A, Miesner M & Wilkerson M 2016 Effect of bovine sperm-bound antisperm antibodies on oviductal binding index. Reprod uction in Dom estic Anim als 51 287293. (https://doi.org/10.1111/rda.12679)

    • Search Google Scholar
    • Export Citation
  • Ferrer MS, Klabnik-Bradford J, Anderson DE, Bullington AC, Palomares RA, Miller LMJ, Stawicki R & Miesner M 2017 Sperm-bound antisperm antibodies prevent capacitation of bovine spermatozoa. Theriogenology 89 5867. (https://doi.org/10.1016/j.theriogenology.2016.10.012)

    • Search Google Scholar
    • Export Citation
  • Fijak M, Iosub R, Schneider E, Linder M, Respondek K, Klug J & Meinhardt A 2005 Identification of immunodominant autoantigens in rat autoimmune orchitis. J ournal of Pathol ogy 207 127138. (https://doi.org/10.1002/path.1828)

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