Review: Evaluation of bull fertility. Functional and molecular approaches.

With the term "assisted reproduction technologies" in modern cattle farming, one could imply the collection of techniques that aim at the optimal use of bovine gametes to produce animals of high genetic value in a time- and cost-efficient manner. The accurate characterisation of sperm quality plays a critical role for the efficiency of several assisted reproduction-related procedures, such as sperm processing, in vitro embryo production and artificial insemination. Bull fertility is ultimately a collective projection of the ability of a series of ejaculates to endure sperm processing stress, and achieve fertilisation of the oocyte and production of a viable and well-developing embryo. In this concept, the assessment of sperm functional and molecular characteristics is key to bull fertility diagnostics and prognostics. Among others, functional features linked to sperm plasma membrane, acrosome and DNA integrity are usually assessed as a measure of the ability of sperm to express the phenotypes that will allow them to maintain their homeostasis and orchestrate-in a strict temporal manner-the course of events that will enable the delivery of their genetic content to the oocyte upon fertilisation. Nevertheless, measures of sperm functionality are not always adequate indicators of bull fertility. Nowadays, advancements in the field of molecular biology have facilitated the profiling of several biomolecules in male gametes. The molecular profiling of bovine sperm offers a deeper insight into the mechanisms underlying sperm physiology and, thus, can reveal novel candidate markers for bull fertility prognosis. In this review, the importance of three organelles (the nucleus, the plasma membrane and the acrosome) for the characterisation of sperm fertilising capacity and bull fertility is discussed at functional and molecular levels. In particular, information about sperm head morphometry, chromatin structure, viability as well as the ability of sperm to capacitate and undergo the acrosome reaction are presented in relation to the cryotolerance of male gametes and bull fertility. Finally, major spermatozoal coding and non-coding RNAs, and proteins that are involved in the above-mentioned aspects of sperm functionality are also summarised.

The sperm chromatin structure assay does not detect alterations in sperm chromatin structure induced by hydrogen peroxide.

The objective of this study was to investigate the effects of hydrogen peroxide (H2O2) on the chromatin structure of sperm. For this purpose, 44 cryopreserved bovine ejaculates were analyzed immediately post-thaw (control sperm, CON S), after 1 h of post-thaw incubation (non-oxidized sperm, NOX S), and after 1 h of post-thaw incubation in different concentrations of H2O2 (OX S; 50, 100 µM, 1000 µM H2O2). Sperm motility was determined using computer-assisted sperm analysis. Sperm plasma membrane and acrosome integrity were assessed by flow cytometry after staining with propidium iodide and fluorescein isothiocyanate-conjugated peanut agglutinin. Chromatin damage was assessed using the sperm chromatin structure assay (SCSA), and DNA damage was evaluated using the single cell gel electrophoresis (Comet) assay. Sperm motility and plasma membrane and acrosome integrity decreased while chromatin damage and DNA damage increased after 1 h of incubation (P < 0.05). The addition of H2O2 adversely affected all parameters (P < 0.05) except for chromatin structure. The role of H2O2 in sperm chromatin damage was investigated by supplementation of 10 IU catalase, which reversed the damage (P < 0.05). Interestingly, the chromatin decondensation induced by 10 mM dithioreitol, evidenced by an increase in chromatin damage in the SCSA (P < 0.05), was reversed by H2O2. In conclusion, H2O2 causes alterations in sperm functional status and DNA integrity. However, our results show that high concentrations of H2O2 can induce chromatin condensation, and thus the use of the SCSA for the assessment of H2O2-induced chromatin damage should be carefully considered.

Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility.

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI.

Effects of sodium pyruvate on viability, synthesis of reactive oxygen species, lipid peroxidation and DNA integrity of cryopreserved bovine sperm.

The aim of this study was to examine effects of sodium pyruvate on viability as well as on synthesis of reactive oxygen species (ROS), lipid peroxidation and DNA integrity of cryopreserved bovine sperm. In each of 23 Simmental AI bulls three ejaculates were collected. In a split sample design ejaculates were diluted by using Triladyl® extender without and with the addition of 5mM sodium pyruvate. Both aliquots were equilibrated for 24h before freezing. Frozen sperm samples were thawed, and examined immediately after thawing (0h) as well as after 3, 6, 12, and 24h incubation at 37°C. The percentages of rapidly motile sperm (RMS), plasma membrane and acrosome intact sperm (PMAI), sperm with a high mitochondrial membrane potential (HMMP), amounts of ROS synthesis (dichlorofluorescein-diacetate (DCFH), CellROX Deep Red Reagent® probe (CellROX)) and lipid peroxidation of sperm (LPO) and percentage of sperm with a high degree of DNA fragmentation (%DFI) were determined. Overall, sperm diluted with the extender containing sodium pyruvate showed higher levels of RMS, PMAI and HMMP, CellROX and lower %DFI values (P<0.001) compared to sperm frozen in the extender without sodium pyruvate. However, there was no effect (P>0.05) of sodium pyruvate on LPO and DCFH. The results of this study show that the addition of sodium pyruvate to the semen extender improved the viability as well as DNA integrity of cryopreserved sperm and did not affect their lipid peroxidation, although it increased the synthesis of some ROS.

Seasonal changes of DNA fragmentation and quality of raw and cold-stored stallion spermatozoa.

In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca+2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca2+ level of sperm.

Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen.

This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P < 0.01), and also the extender affected all parameters except HMMP (P < 0.05). After thawing, all parameters except HMMP immediately after thawing were influenced by the equilibration period (P < 0.001), whereas all parameters except % DFI immediately after thawing were influenced by the extender (P < 0.001). The changes of semen characteristics during 3 hours of incubation were also dependent on the equilibration time and the extender used in all parameters (P < 0.01). In the field study, semen of nine bulls was collected thrice weekly, processed using Triladyl egg yolk extender, and frozen in 0.25 mL straws with 15 × 106 spermatozoa per straw. In total, the nonreturn rates on Day 90 after insemination (NRR90) of 263,816 inseminations in two periods were evaluated. Whereas semen collected on Mondays and Wednesdays was equilibrated for 24 hours in both periods, semen collected on Fridays was equilibrated for 4 hours in period one and equilibrated for 72 hours in period 2. No differences in NRR90 could be found (P > 0.05). In conclusion, extension of the equilibration time from 4 hours to 24-72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field.

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality.

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing-thawing.

Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.

The objective was to examine if there are relationships between alterations in sperm viability, reactive oxygen species (ROS) synthesis, and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 hours at 37 °C, and the other frozen, thawed, and incubated for 24 hours at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 hours of incubation. Progressive motile sperm was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm, sperm with a high mitochondrial membrane potential, sperm showing a high degree of DNA fragmentation (%DFI), and their reactive oxygen species content were assessed with dichlorofluorescein-diacetate, dihydrorhodamine, diaminofluorescein diacetate, and mitochondrial superoxide indicator using flow cytometry. Although all other sperm parameters showed alterations (P < 0.05) during the 24-hour incubation time, %DFI stayed constant (P > 0.05, 0.91 ± 0.23) in nonfrozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, dichlorofluorescein-diacetate-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species.

Chromatin integrity of ram spermatozoa. Relationships to annual fluctuations of scrotal surface temperature and temperature-humidity index.

The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SST mean) and THI (THI mean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SST mean, and THI mean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SST mean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THI mean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SST mean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa.