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- Springer Journals
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- Copyright © The Author(s) 2023
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- 2049-1891
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- 10.1186/s40104-022-00810-3
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Abstract
Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stal‑ lions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. Methods We used frozen‑thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not‑migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor staining with Motic Images Plus 3 software. Additionally, migrated and not‑migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. Results The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not‑migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, char ‑ acterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not‑migrated sperm (χ², P < 0.05). Conclusion Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not ‑selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates. Keywords ART , Bovine, Sperm morphometry, Sperm selection, Thermotaxis *Correspondence: Mistral Fertility Clinics S.L, Clínica Tambre, Calle Tambre, 8, 28002 Madrid, Alfonso Gutiérrez‑Adán Spain agutierr@inia.csic.es Department of Animal Reproduction, INIA‑ CSIC, 28040 Madrid, Spain © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 2 of 11 of the percentage of cleavage can reach up to 80% but Background embryo production remains between 30% and 40% [16, It has been shown that, in mammals, from the millions 17]. In contrast, ICSI is a very inefficient procedure in of spermatozoa ejaculated, only tens to hundreds reach bovine that is only successful when the oocytes are artifi - the site of fertilization at the ampulla [1]. Presumably, cially activated [18] yielding a low number of blastocysts this selected group of spermatozoa have better charac- (about 20%) [19, 20]. This inefficiency can be due to sper - teristics for supporting fertility and embryo development matozoa delayed or incomplete nucleus decondensation [2]. The guidance of the spermatozoa through the female [21]. Even though IVF is widely used, the development genital tract has been proposed to be a physiological phe- of ICSI in bovine could be useful to decrease the genera- nomenon to help them reach the oocyte in vivo [3]. Dif- tion interval, use of high valuable males and use semen ferent guidance mechanisms have been described in vitro samples with low sperm numbers, such as those obtained which are chemotaxis [4], rheotaxis [5] and thermotaxis after sex-sorted [22]. Interestingly, in IVF it is necessary [6]. Sperm thermotaxis is defined as the ability of the to use at least 10,000 spermatozoa per oocyte to have a spermatozoa to move in a temperature gradient from the good cleavage rate [23] suggesting that not all the sper- lower to the higher [6], and it is present in different spe - matozoa are equally competent to fertilize the oocyte. cies such as rabbits, humans [6], mice [7], horses [8] and An approach to overcome the problems of ICSI in also in bull [9]. However, in bull, it has been evaluated the bovine would be to mimic some of the events that would ability of the spermatozoa to sense the gradient but not occur in vivo during fertilization and that are bypassed the quality of the spermatozoa migrated [9]. Consider- with ICSI [24], such as the sperm selection by thermo- ing the presence of this phenomenon in different species, taxis as it is a guidance mechanism known to operate this guidance mechanism could be understood as a phe- in the oviduct [6]. It has been previously described in nomenon conserved among species. human, rabbit and mouse that the spermatozoa have to In mice, humans and horses, the recovery of the sper- be capacitated to be able to sense the temperature gradi- matozoa migrated by thermotaxis has shown lower levels ent [6, 7]. Capacitation involves all those biochemical and of DNA fragmentation than the not-selected ones; while structural changes that spermatozoa must undergo to be in mice, intracytoplasmic sperm injection (ICSI) with able to penetrate an oocyte [25]. Heparin is widely used spermatozoa selected by thermotaxis increased the pro- for bull sperm capacitation in vitro [26]; however, other duction of blastocysts with higher quality and percent- molecules have been used for sperm capacitation in vitro age of live births [7]. Sperm DNA fragmentation (SDF) like caffeine [27, 28], among others [29–31]. has been linked to poor rates of embryonic development Bovine sperm has been reported to sense a tempera- and conception and increased number of miscarriages, ture gradient and move toward the warmer side [9]. morbidity in the offspring and even childhood cancer However, analysis of the quality of the migrated sperma- [10]. Besides, in mice, DNA fragmentation has also been tozoa, and its use in ICSI has not been performed yet. linked to long-term multigenerational consequences and Consequently, this study aimed to evaluate the migra- premature ageing, such as aberrant growth, mesenchymal tion of frozen-thawed bull spermatozoa by thermotaxis tumours, and abnormal behavior [11]. Moreover, we have with the methodology described by Pérez-Cerezales et al. reported that the use of DNA-damaged sperm reduced [7] and establish the conditions of capacitation neces- the rates of preimplantation embryo development and sary for this system [7]. Besides, quality evaluation of the reduced the number of offspring [12]. In addition, dur - migrated spermatozoa was performed, in terms of SDF, ing the passage of the sperm through the epididymis, head morphometry, and subpopulation structure, while the compaction of the chromatin is produced due to the their fertilizing competence through ICSI was assessed. change from histones to protamines, which reduces the size of the sperm head [13]. Therefore, a bigger sperm Methods head area has been associated with deficiencies in chro - Experimental design matin compaction and higher abnormalities [14], which This study aimed to evaluate the ability of frozen-thawed impairs embryo development in bovine. Chromatin com- bull spermatozoa to migrate by the thermotactic system paction and thus, its integrity, could be indirectly evalu- developed by our group and to assess the quality of the ated by analyzing the sperm head morphology (size and spermatozoa recovered according to the DNA integ- shape) [15]. Taken all these together, efficient sperm rity, and size of the sperm head and developmental rates selection mechanisms are needed to select viable and (cleavage rate and blastocyst yield) by ICSI. First, we high-quality spermatozoa. examined whether or not bull spermatozoa were able to In bovine in vitro fertilization (IVF), the most common migrate during 1 h of selection in the thermotactic sys- sperm selection technique is density gradient centrifuga- tem with a temperature gradient from 36 ℃ to 39 ℃, tion (DGC). Using DGC, the IVF success rate in terms Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 3 of 11 using the FERT medium supplemented with 5 mmol/L of thermotaxis was calculated as follows: [100 × (number caffeine and 25 mmol/L of HEPES. Then, the sperm DNA of spermatozoa migrating within the temperature gradi- fragmentation was evaluated using the neutral version of ent (36 ℃ to 39 ℃) − number of spermatozoa migrating the comet assay and the size of the head of the spermato- within the controls (selecting the control, which resulted zoa selected was analysed using the Hemacolor staining. in higher random migration)/number of spermatozoa Finally, the fertilizing competence of the thermotactic loaded]. For the initial setting of the system, all controls selected spermatozoa was tested in vitro by ICSI using were used. For the analysis of DNA fragmentation and in vitro matured oocytes (IVM), without activation. ICSI, the inverted control was the only control settled as it was necessary to recover the maximum spermato- Sperm processing zoa migrated by thermotaxis due to the low numbers Approval from an ethical committee to conduct this study recovered. was not required as all performed experiments were in vitro. Frozen-thawed seminal samples from 6 different DNA fragmentation analysis Asturian Valley bulls with proven fertility were provided DNA fragmentation was analysed employing the neu- by the Regional Service of Agrifood Research and Devel- tral version of the single cell gel electrophoresis assay opment (SERIDA), Gijón, Spain. Animals were selected (SCGE or Comet assay) as previously described with based on their artificial insemination (AI) outcomes some modifications [7]. Briefly, the samples were pelleted using frozen samples being above 50% of the non-return by centrifugation (600 × g) and diluted to a maximum rate (62% ± 9%, n = 6). Four to five straws (0.25 mL) of 20 × 10 spz/mL in 0.5% low melting point agarose in of each bull (same ejaculate) were thawed at 37 ℃ in a PBS. Because of the low numbers obtained in the ther- water bath for 40 s. Motile spermatozoa were selected by motaxis assay, the samples of migrated spermatozoa were BoviPure gradient (Nidacon Laboratories AB, Göthen- accumulated (pull of four capillaries) and used entirely. borg, Sweden) centrifuged for 10 min at 290 × g. The Immediately after dilution, 85 µL were placed on a slide resulted pellet was then resuspended in Boviwash solu- previously coated with 1% agarose and covered with a tion (Nidacon Laboratories AB, Göthenborg, Sweden) 22 mm × 22 mm coverslip. The slides were then left at 4 and centrifuged for 5 min at 290 × g. Finally, the pellet ℃ for 15 min for agarose polymerization. Then, the lysis was resuspended to a final concentration of 10 × 10 spz/ protocol used was the previously described by Ribas- mL in FERT medium [Tyrode’s medium with 25 mmol/L Maynou specifically designed for bull sperm [32]. After bicarbonate, 22 mmol/L sodium lactate, 1 mmol/L lysis, the slides were washed for 30 min in neutral elec- sodium pyruvate, and 6 mg/mL fatty acid-free bovine trophoresis solution (90 mmol/L Tris, 90 mmol/L boric serum albumin (BSA)] supplemented with 10 µg/mL acid, and 2 mmol/L EDTA, pH 8.5) and then subjected of heparin or 5 mmol/L of caffeine and 25 mmol/L of to electrophoresis (25 V, 300 mA, for 10 min). After this, HEPES. the slides were then washed in neutralization solution (0.4 mol/L Tris-HCl, pH 7.4) for 30 min, then washed in Sperm thermotaxis distilled water for 10 min and finally fixed in methanol Sperm thermotaxis was performed as previously for 3 min, air-dried, and stored until analysis. The sam - described by Pérez-Cerezales et al. [7] with some modi- ples were stained with 30 µL of ethidium bromide, cov- fications. Briefly, the medium used for bull sperm selec - ered with a 22 mm × 22 mm coverslip, and subsequently tion was FERT supplemented with 25 mmol/L of HEPES observed in a fluorescence microscope Nikon Optiphot-2 and 5 mmol/L of caffeine. The temperature gradient was (Nikon, Tokyo, Japan). Comets were digitalized with a set up from 36 ℃ to 39 ℃. The spermatozoa were loaded Nikon 5100 digital camera (Nikon, Tokyo, Japan) cou- at a concentration of 10 × 10 spz/mL in the drops at 36 pled to the microscope. From 150 to 200 comets were ℃ (for migration by thermotaxis and for the temperature analysed per sample using the free software Casplab control) and the drops at 39 ℃ (for the inverted control 1.2.3beta2 (CaspLab.com) [33]. and constant control temperature) and they were allowed to migrate for 1 h. After this time, migrated and not- Sperm morphometry evaluation migrated spermatozoa were recovered and processed To evaluate sperm head morphometry (length, width, for SDF, hemacolor staining analysis, and ICSI. The con - perimeter and area of the head) the Hemacolor Rapid trols for random migration were the same as the previ- staining (Merck, Madrid, Spain) was used for the sper- ous study [7] but modified the temperature, therefore matozoa in both migrated and not-migrated groups after the non-gradient controls were at the same temperatures thermotaxis. A total of 5 µL of the not-migrated group (36 ℃ to 36 ℃ and 39 ℃ to 39 ℃) and the inverted con- was deposited on a microscope slide, then smeared and trol was set from 39 ℃ to 36 ℃. The percentage of net left to dry at room temperature (RT). Because of the low Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 4 of 11 Multivariate procedures analysis number of spermatozoa in the migrated group, a 20-µL Multivariate procedures were performed to identify drop was partially smeared in this group and then air- sperm subpopulations according to the morphomet- dried at RT. Hemacolor staining was applied following ric sperm variables. First, the data was standardized to kit recommendations. Slides were stained by immersion avoid any scaling effect. The principal component analy - for 3 min in a fixing solution, 2 min in a colour reagent sis (PCA) was used to reduce the dimensionality of the red containing Eosin Y and 2 min in a colour reagent data, previously testing the adequacy of the data using blue containing azur B. A total of 32 slides were analysed Bartlett’s sphericity test and KMO (Kaiser-Meyer-Olkin) (8 slides from each replicate: 4 slides per experimental test. Then, the principal components with eigenvalue > 1 group). In each slide, 25 spermatozoa with well-formed (Kaiser criterion) were selected to compute the varimax- heads were measured using the Motic BA210 microscope rotated principal components, which will explain a high equipped with a Moticam 3.0 MP CMOS Digital Cam- percentage of the total variance. Afterwards, the non- era. A picture of the spermatozoa was obtained using a hierarchical clustering method k-means was used to 100× lens and the area of the head was measured with classify the spermatozoa by morphology into a reduced the Motic Images Advanced 3.2 software. number of subpopulations. The k-means method uses a Based on the data obtained from the measurement of predefined number of clusters which was selected using the sperm head, calculations were made for the param- the minimum total within-cluster sum of squares. Sperm eters related to the shape of the structure: ellipticity subpopulations were characterized in terms of their mor- (length/width), rugosity (4π × area/perimeter ), elonga- phometric variables. tion [(length − width)/(length + width)] and regularity (π × length × width/area) [34]. Elongation and ellipticity Oocyte collection and in vitro maturation values describe the size of the cell in terms of how long Bovine ovaries were recovered from a local slaughter- (the higher the value, the longer the cell), and how wide house, and immature cumulus-oocyte complexes (COCs) it is (value above 1 refers to an elliptical cell, and a value were obtained by aspirating follicles (2–8 mm) from equal to 1 is indicative of round cells), respectively. The the ovaries of mature heifers and cows. After selection, regularity describes more precisely the format within the COCs with homogeneous cytoplasm and intact cumu- variations of the ellipse. A value equal to 1 indicates the lus cells (grade I and II) were maturated in groups of 50 perfect ellipse. The rugosity characterizes the amorphous COCs per well in four-well dishes (Nunc, Roskilde, Den- cells with a lower value, indicating a rougher surface of mark) containing 500 µL maturation medium (TCM- the head. This parameter can be positively correlated 199), supplemented with 10% fetal calf serum (FCS) and with head length, as well as being indicative of cell sus- 10 ng/mL epidermal growth factor (EGF). Oocytes were ceptibility to damage/rupture [35]. IVM for 24 h at 38.5 °C, with 5% C O in air with maxi- mum humidity. Acrosomal exocytosis analysis The acrosomal exocytosis evaluation was performed as In vitro fertilization (IVF) previously described [36]. Slides were washed for 5 min As a control for the ICSI, we set a parallel standard twice in PBS and 50 µL of 15 µg/mL of fluorescein iso - IVF group using the IVM oocytes from the same batch thiocyanate-conjugated peanut agglutinin (FITC-PNA) and the spermatozoa selected by DGC. As previously (L7381, Merck) and 6.5 µg/mL Hoechst 33,342 in PBS described in sperm processing, frozen semen straws were added and covered with a 24 × 24 cover slide. The from a bull of proven fertility were thawed at 37 ℃ in a slides were then incubated for 30 min at room tem- water bath for 40 s. Sperm selection was performed with perature in a humid box. Subsequently, the slides were Bovipure (Nidacon Laboratories AB, Göthenborg, Swe- washed in distilled water for 10 min and mounted with TM den), and the final sperm concentration was adjusted to Fluoromount aqueous mounting medium (Sigma- 1 × 10 spz/mL for fertilization. Gametes were co-incu- Aldrich, St. Louis, MO, USA), sealed with nail pol- bated in 500 µL fertilization medium (Tyrode’s medium ish and examined in a fluorescence microscope Nikon with 25 mmol/L bicarbonate, 22 mmol/L sodium lactate, Optiphot-2 (Nikon, Tokyo, Japan). At least 200 sperma- 1 mmol/L sodium pyruvate, and 6 mg/mL fatty acid-free tozoa from two slides per sample were analysed by blind BSA) supplemented with 10 µg/mL heparin sodium salt counting using codified slides. Only completely reacted (Calbiochem, San Diego, CA, USA) in a four-well dish, in acrosomes were counted as reacted and spermatozoa groups of 50 COCs, for 18–20 h at 38.5 °C, 5% C O in air showing partial staining in the acrosome were counted as with maximum humidity. unreacted. Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 5 of 11 Intracytoplasmic sperm microinjection (ICSI) spermatozoa subpopulations. Differences were consid - ICSI was performed with Piezo-actuated micromanipu- ered significant when P < 0.05. One-way ANOVA was lation adapted from mouse ICSI protocols, previously performed to analyze the differences between sperm described [37] instead of conventional ICSI with a sharp subpopulations, followed by pairwise t-tests. Statisti- injection needle. ICSI procedure was carried out on a cal significance was considered as P < 0.05. All data were 90-mm Petri dish, with a 50-µL drop of Holding medium analyzed using R (v 4.1.3). (TCM 199, 40% Earle’s Salts (Gibco, 31150-022); 40% Hanks Salts with 25 mmol/L HEPES (Gibco, 22350-029); Results and 20% Fetal Calf Serum) for oocyte manipulation, a Sperm migration by thermotaxis using heparin vs. caffeine 10% PVP in PBS drop for injection needle wash; and as capacitating molecules several 20 µL drops of a mixture 1:5 of 10% PVP in PBS: Because sperm capacitation is a prerequisite to respond Holding medium, covered by mineral oil. Bovine oocytes to thermotaxis, we first used heparin for inducing sperm were injected in groups of 10, alternating among sperm capacitation as it is widely added in the capacitation groups. Sperm was kept in the same medium used in media [26]. The medium used was FERT supplemented thermotaxis, at 38 °C, and 5 µL was placed in a fresh drop with 25 mmol/L of HEPES with heparin at a concentration immediately before its use. Spermatozoon with proper of 10 µg/mL, which is the standard protocol used for bull motility was captured by suction with the injection nee- sperm capacitation. In these conditions, we observed that dle and, after breaking the mid-piece of the flagellum heparin produced a strong head-to-head agglutination, by a piezo pulse to avoid any movement of the sperma- as previously reported [26] which affected sperm motil - tozoon, they were moved to a manipulation drop, and ity and prevented sperm from migrating in response to injected into the oocyte cytoplasm after passing through thermotaxis. Then, we analyzed whether caffeine, which the zona pellucida. Injected oocytes were recovered from has been previously described as a capacitating agent in the manipulation drop and placed in a culture medium frozen-thawed bull spermatozoa [27], could promote for embryo development. Furthermore, a control group capacitation without increasing sperm agglutination. was settled in which the oocytes were injected with the We supplemented the FERT medium with 25 mmol/L of needle without spermatozoa (sham group). HEPES and 5 mmol/L of caffeine for sperm migration by Injected and control oocytes were cultured in groups of thermotaxis and we found that this concentration, which 20–25 zygotes in 25 µL droplets of synthetic oviduct fluid has been reported that induced capacitation in bovine (SOF) [38] with 4.2 mmol/L sodium lactate, 0.73 mmol/L spermatozoa [39], avoided sperm head-to-head agglutina- sodium pyruvate, 30 µL/mL BME amino acids, 10 µL/mL tion, therefore, it was the medium of choice. MEM amino acids, 1 µg/mL phenol-red and 5% FCS When thermotaxis was performed under these condi- (F2442, Sigma). Approximately 18–20 h post-insemina- tions, we obtained a higher number of sperm migrated tion (hpi), presumptive zygotes from the control group in the thermotactic unit (from 36 ℃ to 39 ℃) than the were denuded of cumulus cells by vortexing for 3 min rest of the controls (P < 0.006, n = 6) (Fig . 1 A). This and then cultured in groups of 20–25 in 25 µL droplets of means that, a higher number of spermatozoa migrated SOF covered with mineral oil at 38.5 °C, 5% CO , 5% O by sensing the gradient, from the lower temperature (36 2 2 and 90% N . Cleavage rate was recorded on day 2 (48 hpi) ℃) to the higher temperature (39 ℃), than the random and blastocyst yield on day 7 post-insemination. A total migration obtained in the controls of constant tempera- of 8 replicates were used to assess embryo development ture (at 36 ℃ and 39 ℃) and the inverted control (migra- after ICSI. tion from 39 ℃ to 36 ℃). The average of the total number of cells migrated by thermotaxis was 4.3 × 10 spz and the percentage of net thermotaxis obtained with this Statistical analysis medium was 3.6% (n = 6). We did not find differences Data were analyzed by descriptive statistics based on between the number of sperm that migrated randomly the mean ± standard deviation calculated for each of the in the three controls established (controls of constant variables. Differences among treatments were analyzed temperatures at 36 ℃ and 39 ℃ and the inverted control using one-way ANOVA, and Post-hoc analysis to iden- from 39 ℃ to 36 ℃). tify differences between groups was performed using Tukey test for parametric analysis or Kruskal-Wallis’s Eec ff t of sperm thermotaxis on DNA integrity test for non-parametric analysis. A Chi-square test (χ²) We next examined, by neutral COMET assay, if the sper- was used to analyze the differences between samples. matozoa migrated by thermotaxis had higher DNA integ- A Z-test was used for the analysis of the percentage of rity. We obtained a lower SDF level in the spermatozoa acrosome-reacted spermatozoa and percentages of migrated by thermotaxis (from 36 to 39 ℃) in comparison Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 6 of 11 Fig. 1 Number, DNA fragmentation, and head size of the spermatozoa selected by thermotaxis. A Number of spermatozoa selected by thermotaxis with the medium supplemented with 5 mmol/L of caffeine in the thermotactic migration unit, thermotaxis ( Tx) group (36 to 39 ℃), the inverted control (39 to 36 ℃) and the constant control temperatures (36 to 36 ℃ and 39 to 39 ℃). The asterisk indicates significant differences between groups (Kruskal‑ Wallis, P < 0.05). The initial number of spermatozoa loaded in the thermotaxis system was 10 × 10 spz/mL per separation unit, n = 6. B Percentage of DNA fragmentation in the spermatozoa selected by thermotaxis ( Tx) and the not‑migrated spermatozoa (NM), n = 8. Asterisk indicates significant differences between groups. C Area of the head size from spermatozoa. Number of spermatozoa migrated by thermotaxis ( Tx) and not‑migrated (NM) (4 repetitions; n = 400 spermatozoa analyzed for repetition). χ², P < 0.01. D Representation of the distribution of the head area value of spermatozoa migrated by thermotaxis ( Tx) and not‑migrated (NM) to the spermatozoa not-migrated, that remained in the Table 1 Mean values (± SD) of each morphometric parameter thermotactic separation unit (the remaining spermatozoa corresponding to bull sperm migrated or not migrated by in the drop at 36 ℃ that was not able to migrate to the thermotaxis drop at 39 ℃) (Fig. 1B) (P < 0.001, χ², n = 8). The obtained Item Tx NM percentage of reduction in the DNA fragmentation was ** 13.3% between groups. Length, µm 9.14 ± 0.38 9.24 ± 0.36 ** Width, µm 4.17 ± 0.22 4.24 ± 0.23 *** Sperm head morphometry differences Perimeter, µm 32.45 ± 1.79 34.15 ± 2.03 2 *** The results of the comparison of the morphometric Area, µm 37.83 ± 2.20 38.80 ± 1.82 dimensions of sperm heads from spermatozoa migrated Ellipticity, L/W 2.20 ± 0.15 2.19 ± 0.15 or not migrated by thermotaxis are summarized in Rugosity, 4πA/P 0.46 ± 0.06 0.42 ± 0.05 Table 1. A total of 400 properly digitalized spermatozoa Elongation, (L‑ W )/(L + W) 0.37 ± 0.03 0.37 ± 0.03 were analyzed (4 replicates). Kruskal-Wallis one-way Regularity, πLW/4A 0.79 ± 0.07 0.79 ± 0.06 analysis of variance on ranks showed a significant effect SD Standard deviation. Significant differences between Tx and NM by Kruskal‑ ** *** of migration by thermotaxis on sperm head morphom- Wallis one‑ way analysis of variance on ranks, P < 0.01, P < 0.001 etry. The four sperm-head parameters of size were sig - nificantly different; and of the shape parameters, only rugosity was different (Table 1; Fig. S1). Sperm that length, width, and ellipticity, suggesting that within the responded to thermotaxis had a smaller head size than population of spermatozoa that migrate by thermotaxis, sperm that do not respond (Fig. 1C,D). To eliminate the there may also be two subpopulations, one that could cor- possibility that the sperm that migrate by thermotaxis respond to the once migrated correctly because responded have undergone the acrosome reaction and have there- to thermotaxis (a population that is different from non- fore smaller head area, we analyzed the presence of the migrated sperm) (Fig. 1D), while the other (population acrosome employing the fluorescent probes PNA-FITC with a peak similar to the control) could be sperm that [36] and found that in both cases the percentage that have migrated by chance, as it happens with the spermato- had completely lost the acrosome were similar (1.25% vs. zoa that migrate by chance in the control groups (Fig. 1 A). 3.75% for not-migrated and migrated respectively). Interestingly, the head area distribution in thermot- Sperm head subpopulation differences active-responsive sperm had two peaks while the not- The principal component (PC) analysis of the sperm head migrated group only had one (Fig. 1D). The same effect morphometry data from both spermatozoa migrated was observed in other parameters measured such as Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 7 of 11 and not-migrated by thermotaxis, produced three PCs, explaining 89.1% of the variance (Table S1). PC1 was represented by length, width, ellipticity, and elongation components; PC2 was represented by perimeter and rugosity, and PC3 was represented by area and regularity. The analysis of subpopulations revealed four well-defined groupings (Fig. S2). In Fig. 2 and Table S2, we can see the mean values of the morphometric parameter cor- responding to the 4 subpopulations (SP) and the signifi - cant differences between SPs (Fig. S3). The characteristics of SP1 showed the highest PC2, SP2 showed the lowest PC2, SP3 showed the lowest PC1 and SP4 showed the highest PC1. The spermatozoa from SP1 have high length, width, Fig. 3 Rate of cleavage and blastocyst production. Percentage of perimeter and regularity values, and this SP1 is reduced cleavage and blastocyst production of the embryos obtained by ICSI using the spermatozoa migrated by thermotaxis ( Tx) (n = 8, P < 0.01 in the thermotactic group (Fig. 2). SP2 has low length, for cleavage), not‑migrated (NM) by thermotaxis (n = 8), obtained by width, perimeter and regularity, but has high ellipticity conventional IVF (n = 6) and oocytes microinjected without sperm and elongation, and higher rugosity value (poorly amor- (Parthenogenetic) (n = 4). Different letters above error bars (i.e. ±SEM) phous and rough cells). This subpopulation is the one indicate significant differences (P < 0.001) among groups ( Tukey’s that increases the most in the thermotactic group (Fig. 2). posthoc test) No differences were found for SP3 (higher width and lower elongation and regularity) and SP4 (higher length and higher ellipticity and elongation values). The results higher cleavage rate in comparison to those microin- indicate that the spermatozoa that have migrated by ther- jected with not-migrated spermatozoa and also with motaxis have small length, width, and perimeter but are the sham group (oocytes microinjected without sperm elongated in an elliptical shape and with very little rough- (26.2% ± 1.7% vs. 9.3% ± 2.7% vs. 7.2% ± 3.1% respec- ness in their membrane, which corresponds to the SP2. tively, P < 0.001) (Fig. 3). When looking at the blastocyst production (percentage of blastocyst out of the cleaved ICSI with migrated spermatozoa embryos) a significantly higher production was obtained The results obtained after ICSI, employing IVM oocytes when it was used migrated spermatozoa compared with with sperm selected by thermotaxis showed a significant those oocytes microinjected with the not-migrated Fig. 2 Subpopulation distribution of bull sperm selected by thermotaxis. A Scaled mean values of the 8 morphometric variables in the four sperm subpopulations. B Proportion of spermatozoa belonging to each subpopulation in the spermatozoa migrated by thermotaxis, and (C) in the not‑migrated spermatozoa Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 8 of 11 sperm and the sham group (considered a partheno- medium according to the species-specific necessities genetic division) (32.2% ± 9.3% vs. 8.3% ± 7.8% and 0 or the origin of the semen sample used, as frozen sam- respectively, P < 0.05). The IVF control (IVM oocytes and ples (like the ones used for horse) gave a higher migra- spermatozoa selected only by DGC) gave a cleavage rate tion percentage than the fresh (mouse and human) [7, of 84.1% ± 1.9% and a blastocyst yield of 35.5% ± 8.3% 8]. Should be taken into consideration that the frozen- demonstrating that the oocytes were properly matured thawed sperm show capacitation-like events due to and the semen sample was adequate. the procedure of freezing and thawing itself, known as cryocapacitation [41] and, as a consequence of this, a certain degree of capacitation is obtained right after Discussion thawing. This is the reason why the thermotactic assay This study aimed to evaluate bull sperm selection by was performed immediately after DGC. thermotaxis and the quality of the spermatozoa migrated Sperm selected by thermotaxis has lower DNA frag- according to their DNA fragmentation, sperm head mentation index than not-selected sperm. DNA frag- morphometry, and their ability to fertilize IVM oocytes mentation is one of the parameters whose analysis has through ICSI. Here, we report the successful migration gained great importance for the prediction of ART suc- by thermotaxis of frozen-thawed bull sperm using the cess [42]. Sperm DNA damage induces fragmentation of system previously developed by Pérez-Cerezales et al. chromosomes and segregation errors leading to mosai- [7]. Furthermore, to our knowledge, this is the first time cism of embryos [43] compromising embryonic develop- that the quality of bull sperm migrated by thermotaxis ment [44]. In this study, we found a reduction of 13.3% of is evaluated showing higher DNA integrity and lower the levels of DNA fragmentation in the sperm migrated sperm head size. Moreover, when used for ICSI (without by thermotaxis in comparison to the control confirming oocyte activation) we were able to obtain a higher cleav- that we could select, with this methodology, a sperm sub- age and blastocyst formation rates compared with the population with higher quality in terms of DNA integ- not-migrated group. rity as previously reported in other species [7, 8]. Taking We applied here the system developed by Pérez- into account that the conventional sperm selection tech- Cerezales et al. [7] as it can recover mice and human niques (swim-up and DGC) have not been able to dem- spermatozoa with high quality and in the case of mice, onstrate the selection of spermatozoa with lower SDF improve ICSI outcomes. Consequently, in this study, [45], the results reported here suggest that thermotaxis we adapted the temperature of sperm selection for could be a useful sperm selection technique to recover bulls with respect to mice, humans and horses [7, 8] the sperm subpopulation with better quality in terms of as the gradient established here went from 36 to 39 DNA integrity. ℃ instead of 35 to 38 ℃. This change was made tak - Another sperm quality parameter analyzed was sperm ing into consideration the normal core body tempera- head morphology as it has been linked to the sperm ture of a healthy cow, and the temperature of incubation nuclear compaction and chromatin integrity [15]. A big- in bovine IVF which is 38.5 ℃, therefore, this gradient ger sperm head area has been associated with deficien - would be the most suitable for bull sperm selection. The cies in protamine compaction and higher abnormalities medium used in this study for migration by thermotaxis [14], while thinner sperm nuclei are related to higher fer- was the FERT, supplemented with 5 mmol/L of caffeine tilization rates in mice [14, 46]. Also, differences in sperm instead of heparin to avoid sperm head-to-head agglu- head length have been directly related to conception tination. In these conditions, migration by thermotaxis rates in other species. For example, an increase in the was achieved in bull sperm, obtaining a net thermotaxis coefficient of variation of the sperm head length in bulls of 3.6% which is higher than that obtained for stallion and stallions has been related to a reduction in fertility (1.1%), mouse (0.5%) and human (0.8%) using the same [47, 48]. Also, in red deer and ram, it has been reported sperm selection system [7, 8]. The spermatozoa have to that males with high fertility rates have ejaculates with a be capacitated to migrate by thermotaxis [6] and under larger proportion of spermatozoa having small and elon- capacitating conditions, we know that only about 10% gated heads [49, 50]. In agreement with these, our results of them are capacitated at a given point [40]. There - demonstrated that spermatozoa migrated by thermotaxis fore, it could be expected that the percentage of sper- had a smaller sperm head size and higher elongation and matozoa able to migrate by thermotaxis would be lower ellipticity values in comparison to the not-migrated sper- than 10%, as previously reported for different species matozoa. Thus, the reduction of the head size and elon - [7, 8] and also confirmed with current results. Never - gation of the spermatozoa migrated by thermotaxis could theless, the net thermotaxis was higher for bull sperm be related to the selection of spermatozoa with higher in comparison to the species aforementioned, which quality. could be due to the adequation of the capacitation Ruiz‑Díaz et al. Journal of Animal Science and Biotechnology (2023) 14:11 Page 9 of 11 Finally, to test the quality of the bull spermatozoa Supplementary Information migrated by thermotaxis, we performed ICSI, which is The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40104‑ 022‑ 00810‑3. an inefficient procedure in bovine that is only successful when the oocyte is artificially activated [18]. We reported Additional file 1: Table S1. Eigenvalues of each parameter in the three here that, without oocyte artificial activation, the percent - PCs for bull sperm head morphometry found in sperm migrated or not age of cleavage rate and blastocyst yield were higher for migrated by thermotaxis. those oocytes microinjected with spermatozoa selected by Additional file 2: Table S2. Mean values (± S.D.) of each morphometric parameter corresponding to different SPs from bull sperm head morpho ‑ thermotaxis vs. those not-selected, as well as previously metry found in sperm migrated or not migrated by thermotaxis. reported for mice [7]. Some of the causes of the inefficiency Additional file 3: Fig. S1. Density plots showing the distribution of the 8 of ICSI in bovine are failure of the oocyte activation after morphometric variables in both migrated and not‑migrated spermatozoa microinjection and defective sperm head decondensation after thermotaxis. [19, 21, 51]. These results suggest that thermotaxis might Additional file 4: Fig. S2. Distribution of sperm subpopulations accord‑ have been able to select spermatozoa with the ability to ing to their PC values. activate the oocyte or decondense the sperm head properly Additional file 5: Fig. S3. Boxplots showing the distribution of the morphometric variables of the four subpopulations along with mean after cytoplasmic injection. We hypothesized that sperma- comparison P‑ values obtained by the t‑test. tozoa selected by thermotaxis could be properly capaci- tated and this could be the reason why oocyte activation Acknowledgements was not necessary to achieve similar or even higher embryo This manuscript is dedicated to the memory of our colleague, Dr Serafín Pérez production rates in comparison to other studies that used Cerezales, who recently passed away. We thanks to Dr. Michael Eisenbach for ICSI with oocyte activation [19, 20, 51]. These results are reviewing the manuscript. prominent for future studies to optimize the current meth- Authors’ contributions odology for bull sperm selection by thermotaxis, which Experimental work: SRD, RM, CdF, RFG, MM, PB, CC, ML. Data analysis: SRD, PNL, in combination with ICSI and oocyte activation might DR and AGA; Interpretation of data: SRD, PNL, DR and AGA; Funding acquisi‑ tion: DR and AGA; Literature review: SRD, DR and AGA; Conceptualization: increase the success of ICSI in this species. AGA and DR. Writing the original draft: SRD, RM, RFG, CdF, DR and AGA. All the authors reviewed and approved the final version of the manuscript. Conclusion Authors information Not applicable. Our data showed that bull sperm selection by thermot- axis was achieved using frozen-thawed semen and that Funding the population selected had higher DNA integrity and Open Access funding provided thanks to the CRUE‑ CSIC agreement with Springer Nature. This research was funded by the Spanish Ministry of Science lower sperm head size that the not-selected sperma- and Innovation‑MCIN (RTI2018‑093548‑B‑I00 and PID2021‑122507OB‑I00 to tozoa. Besides, when using this population to perform A. Gutierrez‑Adan and PID2019‑111641RB‑I00 to D. Rizos, funded by MCIN/ ICSI, higher cleavage and blastocyst formation rates were AEI/10.13039/501100011033/ and European Union “NextGenerationEU”/PRTR). M.L. was supported by a Juan de la Cierva postdoctoral contract (FJC2019‑ obtained in comparison to the not-migrated spermato- 040385‐I) from the MCIN. S.R.D. was supported by a “Doctorados Industriales zoa. As far as we know, this is the first report showing the 2018” fellowship of Comunidad de Madrid (IND2018/BIO‐9610). R.M. and P.N.L. increased quality of bull sperm migrated by thermotaxis were supported by FPI scholarships from the MCIN (PRE2020‑094452 and PRE2019‑088813 respectively). and its successful use for ICSI, which could make sperm selection by thermotaxis an advantageous technique to Availability of data and materials improve ICSI outcomes in bovine. Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. Abbreviations Declarations AI Artificial insemination BSA Bovine serum albumin Ethics approval and consent to participate COCs Cumulus‑oocyte complex Not applicable. DGC Density gradient centrifugation EGF Epidermal growth factor Consent for publication FCS Fetal calf serum Not applicable. HS Hemacolor staining ICSI Intracytoplasmic sperm injection Competing interests IVF In vitro fertilization The authors declare that they have no competing interests. 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Journal
Journal of Animal Science and Biotechnology – Springer Journals
Published: Jan 11, 2023
Keywords: ART; Bovine; Sperm morphometry; Sperm selection; Thermotaxis