Ethical approval
All veterinary procedures were conducted after receiving the agreement of the Local Ethical Committee for Animal Experiments in Olsztyn (Poland, Agreement Number 7/2019). All experiments were performed in accordance with ARRIVE guidelines, the methods were carried out in accordance with the relevant guidelines and regulations. Moreover informed consent was obtained from the farm owner for the collection of samples.
Animals
The present study was conducted on red deer (hybrids of various subspecies with a predominance of European deer), and it involved mature males (n = 11; 4-5 years; weighing about 145 kg) and mature females (n = 36; 3-4 years; weighing about 95 kg) kept in a deer farm in Rudzie near Gołdap (north-eastern Poland). In spring and fall, the animals were fed only a pasture-based diet, and in winter, their diet was supplemented with silage and cereal grain (10 kg of silage/day/animal + 1 kg of cereal grain).
Collection and initial evaluation of epididymal spermatozoa
Spermatozoa were obtained from the epididymides of 11 stags that were legally culled during the rutting season (September). Sperm samples were collected directly (up to 0.5-1 h post mortem) from the cauda epididymis into Eppendorf tubes, according to the method described earlier by Dziekońska et al. [14]. The samples were assessed for motility and concentration. Sperm motility was determined subjectively. Samples with sperm motility higher than 70% were used in further analyses. The concentration of spermatozoa was determined in a Bürker counting chamber (Equimed-Medical Instruments, Kraków, Poland).
Liquid storage of sperm
Sperm samples were diluted with Bovidyl extender (Bovidyl® (Minitiib GmbH, Germany) containing TRIS, BSA, citric acid, sugars, buffers, proprietary components, antibiotics, double distilled water and 10% clarified egg yolk at room temperature. The samples (100 × 106 spermatozoa/mL) were placed in 50 mL plastic tubes, immediately transferred to a portable refrigerator (5 °C) and transported to the laboratory of the Department of Animal Biochemistry and Biotechnology of the University of Warmia and Mazury in Olsztyn. Sperm samples were stored at a temperature of 5 °C throughout the experiment (11 days).
Experiment 1. Sperm quality analysis
Sperm quality was analyzed on the day the samples were transported to the laboratory (D1) and then on days 5 (D5), 7 (D7), 10 (D10) and 11 (D11).
Analysis of sperm motility parameters
Sperm motility parameters were assessed using the computer-assisted sperm analysis (CASA) system (Hamilton-Thorne Research, HTR, IVOS version 12.3; Beverley, MA, USA). The IVOS settings were applied according to the Hamilton Thorne technical guide v. 12.3 for gazelle/deer [14]. Sperm samples (100 × 106 spermatozoa/mL) were diluted 1:4 with phosphate buffered saline (PBS) and incubated at 37 °C for 20 min. Then the samples (5 μL) were placed in a pre-warmed Makler counting chamber (Sefi-Medical Instruments Ltd., Israel) and examined at 37 °C. A minimum five fields per sample were assessed, with approximately 200 spermatozoa per field. The CASA-determined sperm motility parameters included total motility (TMOT, %), progressive motility (PMOT, %), curvilinear line velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), linearity coefficient (LIN, %) and straightness (STR, %). The spermatozoa were considered motile with VAP >21.9 and VSL > 6.0 μm/s, and progressive with VAP > 75.0 μm/s and STR > 80%.
Sperm morphology assay
Smears were prepared from sperm samples diluted with PBS (1:4). The smears were air-dried and stained by the Giemsa staining method according to Watson [36]. The morphological features of 200 sperm cells from each sample were evaluated under a phase-contrast microscope (1000 × magnification). The percentage of sperm with normal morphology (%, MOR), the percentage of sperm with normal apical ridge acrosomes (%, NAR), and the percentage of sperm with morphological defects of the head, midpiece and tail (%) were determined.
Fluorescence assays
The acrosomal status of spermatozoa was assessed by the fluorescence method using FITC-labeled peanut agglutinin (FITC-PNA; Sigma, Saint Luis, Missouri, USA), as described in a previous study [14] with some modifications. Sperm samples (20 μL, 100 × 106 spermatozoa/mL) were extended in 180 μL of PBS solution and incubated with 2 μL of FITC-PNA solution (2 mg FITC-PNA in 1 mL PBS) at 37 °C for 5 min. Then, 2 μL of propidium iodide solution (PI, 2.4 μM in Tyrode's salt solution) was added to the samples and incubated at 37°C for 5 min. Finally, 10 μL of 10% formalin solution was added to the stained cells. The samples were evaluated under a fluorescence microscope (Olympus BX 41, Tokyo, Japan) at 600× magnification. A minimum of 200 cells per slide were examined in each aliquot. The results were expressed as the percentage of viable sperm cells with intact acrosomes (non-stained spermatozoa in the head region; FITC-PNA-/PI-).
The plasma membrane integrity of sperm was assessed using dual fluorescent probes, SYBR-14 and propidium iodide, PI (Live/Dead Sperm Viability Kit; Molecular Probes, Eugene, OR, USA), according to the method described previously by Garner and Johnson [37] with some modifications. Sperm samples (20 μL, 100 × 106 spermatozoa/mL) were extended in 180 μL of PBS solution. Subsequently, the samples were incubated with 2 μL 1 mM SYBR-14 solution in HEPES-BSA solution (pH 7.4) and 2 μL PI (2.4 μM in Tyrode’s salt solution) at 37°C for 10 min. Aliquots of the stained sperm cells were examined using a fluorescence microscope (Olympus BX 41, Tokyo, Japan) at 600× magnification. A minimum of 200 spermatozoa were counted per slide. The results were expressed as the percentage of viable sperm cells (sperm with bright green fluorescence; SYBR+/PI-).
The mitochondrial membrane potential of sperm was assessed using JC-1 (Molecular Probes, Eugene, USA)/ PI dual fluorescent probes, according to a previously described method [14] with some modification. Briefly, sperm samples (20 μL, 100 × 106 spermatozoa/ml) were extended in 180 μL of PBS solution and incubated with 1 μL of JC-1 solution (1 mg JC-1/mL anhydrous dimethyl sulfoxide, DMSO) for 15 min at 37 °C. Then, sperm samples were stained with 2 μL PI for 5 min at 37 °C. Sperm cells displaying orange-red fluorescence in the mid-piece region were considered as viable spermatozoa with high MMP, whereas sperm cells displaying red fluorescence in the head and green fluorescence in the mid-piece region were classified non-viable spermatozoa with low MMP. A minimum of 200 cells per slide were evaluated using a fluorescence microscope (Olympus BX 41, Tokyo, Japan) at 600× magnification. The results were expressed as the percentage of viable sperm cells with high MMP.
Apoptotic changes in sperm were assessed with the Vybrant Apoptosis Assay Kit #4 (Molecular Probes Inc., Eugene, USA), according to the method described by Trzcińska and Bryła [38] with some modifications. A 200 μL sperm sample (10 × 106 sperm/mL) was first combined with 2 μL JC-1 (100 μM) to facilitate the identification of sperm not stained with YO-PRO-1 or PI, and incubated for 10 min. at 37 °C. Then, 2 µL of YO-PRO-1 (100 µM) and 2 µL of PI (2 µM) were added and incubated again for 10 min. at 37 °C. Aliquots of stained sperm cells were examined under a fluorescence microscope (Olympus BX 41, Tokyo, Japan) at 600 × magnification. A minimum of 200 cells per slide were examined in each aliquot, and four subpopulations were identified in the assay (Fig. 1. A, B): viable sperm categorized as negative for both YO-PRO-1 and PI and positive for JC-1 (YOPRO−/PI−); sperm with apoptotic-like changes in the plasma membrane were categorized as positive for YO-PRO-1 and JC-1, but negative for PI (YOPRO+/PI−); moribund/dead sperm were positive for YO-PRO-1, PI and JC-1 and dead sperm were positive for PI.
DNA integrity was assessed with acridine orange (AO; Life Technologies Ltd., Grand Island, NY, USA), according to the method described by Partyka et al. [39] with some modifications.A suspension of sperm samples (100 μL) was subjected to brief acid denaturation with 200 μL of a lysis solution (Triton X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4). The samples were left for 30 seconds in the dark, and 600 µL of AO solution (6 µg AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6) was added. The samples were left in the dark for 3 min., after which a minimum of 200 sperm cells were randomly selected under a fluorescence microscope (Olympus BX 41) at 600 × magnification. In the analysis, the double-stranded sperm subpopulation (%, DNA integrity) with green fluorescence was distinguished from the sperm population with damaged DNA and yellow to red fluorescence (moderately and completely denatured DNA, respectively).
Experiment 2. Assessment of the fertilizing potential of epididymal spermatozoa
The fertilizing potential of epididymal spermatozoa stored for one, seven and ten days in Bovidyl extender was determined by IVF of the oocytes collected from hinds and by AI of hinds. Sperm samples collected from five males were used for IVF. The samples for AI were pooled from the same males. The samples collected from each male were assessed for quality according to a previously described procedure. The motility of sperm was assessed subjectively before IVF and AI procedures.
The hinds used in both procedures were pharmacologically treated for induction of estrus and ovulation according to the method described by Korzekwa et al. [31].
Induction of Estrus and Ovulation and Oocyte Collection
Ovaries were collected post mortem from 18 red deer hinds (from three groups of 6 hinds each) directly after slaughter at a deer farm in Rudzie near Gołdap (north-eastern Poland) on the 4th day of the estrous cycle after pharmacological synchronization of the animals. Estrus and ovulation were induced during the estrous cycle (September) by applying a single controlled-release vaginal sponge for sheep (Chronogest, MSD Animal Health, Milton Keynes, UK, 20 mg flugestone acetate). For better synchronization, the sponge was removed after 14 days, and equine serum gonadotropin (eCG, formerly known as PMSG; Syncrostim, Ceva Animal Health, Poland, 250 IU) was injected intramuscularly [31]. Estrus was observed 54–56 h after the injection. The day of the estrous cycle was determined based on macroscopic observations of the ovaries and the uterus, and it was confirmed by measuring plasma 17-beta estradiol (E2) and progesterone (P4) levels by a radioimmunoassay (data not shown). Blood was sampled from the heart. The farmed animals were culled for financial reasons and to renew the herd. The ovaries were transported to the laboratory in sterile phosphate buffered saline. Cumulus-oocyte complexes (COCs) were obtained by aspiration from subordinate ovarian follicles measuring less than 5 mm in diameter, and by maceration of ovarian tissue from the same ovary after aspiration. A stereo microscope (Discovery V20, Zeiss, Poznan, Poland) was used to identify COCs consisting of oocytes with homogeneous ooplasm without dark spots, surrounded by at least three layers of compact cumulus cells. The COCs were washed twice in a wash medium (61008; IVF Bioscience, Falmouth, U.K.) and, subsequently, in a maturation medium (61002; IVF Bioscience).
In vitro oocyte maturation and in vitro fertilization
In vitro oocyte maturation (IVM) and in vitro fertilization were conducted according to method described by Korzekwa et al. [31]. Groups of 20 immature COCs were placed in Petri dishes containing 100 µL of the maturation medium covered with mineral oil (M8410, Sigma) and were incubated at 38.5 °C in a 5% CO2 humidified air atmosphere for 23 h. The COCs were washed in a fertilization medium (61003; IVF Bioscience, U.K.).
The COCs were divided into three experimental groups: group 1 was fertilized with the sperm stored in a liquid state at 5 °C for one day (n = 6), and group 2 was fertilized with the sperm stored in a liquid state at 5 °C for seven days (n = 6) and group 3 was fertilized with the sperm stored in a liquid state at 5 °C for ten days (n = 6). Before IVF, sperm was incubated in a capacitation medium (61004; IVF Bioscience, U.K.) for 24 h, and motile spermatozoa were recovered by the swim-up procedure. After incubation, sperm was double centrifuged at 200 × g for 5 min, the supernatant was removed, and the sperm pellet was diluted in an appropriate volume of the fertilization medium to a final concentration of 2 × 106 motile sperm/mL. Groups of 20 COCs were co-incubated with spermatozoa in Petri dishes containing 100 µL of the fertilization medium covered with mineral oil for 15 h at 38.5°C in a 5% CO2 humidified air atmosphere. Embryos (blastocysts) were collected between day 6 and day 9. The developmental stage and quality of the embryos were determined under a microscope (Zeiss Axio Observer A1, Germany; magnification 200×) based on the International Embryo Transfer Society manual. The quality of blastocysts was scored as follows: grade A - excellent; grade B - good; grade C - fair and moderate, grade D - poor and grade E - dead or degenerating (Fig. 2). Embryos classified into quality grades A–C were used to determine the rate of embryo development to the blastocyst stage (IVF success rate) and were assessed for morphological quality.
Artificial insemination (AI) procedure
The AI procedure involved 18 hinds (three groups of 6 hinds each) whose estrous cycle had been previously synchronized (as described above). The first group was inseminated with sperm stored for one day, the second group – with sperm stored for seven days and the third group – with sperm stored for ten days. During the AI procedure, hinds were physically immobilized in a squeeze chute without anesthesia. The hinds were inseminated twice: 56 h after the administration of eCG and then 12 h later (using sperm stored for 2, 8 and 11 days, respectively). The vulvar region was aseptically prepared, and a catheter for intracervical insemination (IMV Technologies, France) was introduced into the cervix. A digital rectal examination was performed to facilitate catheter placement. Around 250 µl of the sperm suspension (containing around 25 - 30 × 106 of spermatozoa) heated to 35–37 °C was placed in a syringe, injected into the catheter and deposited in the cervix. The success rate of fertilization with epididymal sperm was determined by dividing the number of pregnant hinds by the number of inseminated hinds, and it was expressed as percentage. Additionally, the birth rate for hinds was calculated by dividing the number of calves by the number of inseminated hinds. The results were expressed as a percentage.
Diagnosis of pregnancy
Thirty days after insemination, peripheral blood was sampled from the jugular vein to determine the levels of pregnancy-associated glycoproteins (PAGs) and progesterone (P4). PAGs and P4 concentrations were determined according to the method described by Korzekwa et al. [31].
Serum levels of PAGs were determined with the EIA Pregnancy Test (99-41169, IDEXX, Westbrook, ME, USA) after validation. The average intra- and inter-assay coefficients of variation (CVs) were determined at 8.0% and 7.9%, respectively. The presence or absence of PAGs in each sample was validated with the use of the provided formula. The circulating concentrations of PAGs were calculated based on absorbance, where absorbency below 0.3 was indicative of nonpregnancy and absorbance above 0.3 was indicative of pregnancy.
Plasma P4 levels were determined by a radioimmunoassay (RIA) (KIP1458, DIAsource ImmunoAssays, Louvain-la-Neuve, Belgium). The standard curve range was 0.12 to 36 ng/mL, and the ED50 of the assay was 0.06 ng/mL. The intra- and inter-assay CVs were 6.5% and 8.6%, respectively. Hinds with progesterone concentration of 6 ng/mL and higher were considered pregnant [42].
Statistical analysis
Data were examined by repeated measure ANOVA, using the general linear model (GLM) procedure from Statistica software package, version 12.5 (StatSoft Incorporation, Tulsa OK., USA). Shapiro-Wilk and Levene's tests were applied to check the normality of data distribution and the homogeneity of variance, respectively. Data were not normally distributed, and they were transformed accordingly. Percentage data were subjected to arcsine transformation, and motility parameters (VCL, VSL, VAP, ALH and BCF) were log-transformed to obtain normally distributed data before performing one-way analysis of variance (ANOVA). The results were expressed as the mean ± SEM. Significant differences between means were determined by Student Newman–Keuls post hoc test at a significance level of P < 0.05.
Fertilized oocytes, cleavage and blastocyst rates (%), pregnancy rates (%) were analyzed by the Fisher's exact test. Differences were considered statistically significant at P < 0.05.
Data availability
The datasets generated and analyzed during the current study can be made available upon request.