**3. Correlation between** *in vitro* **sperm characteristics and** *in vivo* **bull fertility**

Nowadays, many classical and modern methods have been used for laboratory assessment of *in vitro* semen characteristics following cryopreservation with the main purpose of predicting the fertility potential of a semen sample [11, 31-39].

Among the several sperm characteristics evaluated by laboratory techniques, sperm motility [33,40,41], morphology [42,43] and plasma membrane integrity [11,35,36,38] are the most used laboratory tests for assessing *in vitro* semen quality. However, the results of such assays do not always correlate with the real fertility of a semen sample [12,44].

In this sense, the relationship of *in vitro* semen characteristics and *in vivo* sire fertility has been the subject of much study [12,41,44,45-47]. Nevertheless, substantial variations are commonly observed in different experiments and low correlations are usually detected when single *in vitro* sperm characteristics are isolated compared to the field fertility [12,44]. Until now, the most efficient and accurate method to estimate the fertility of a particular bull is to accomplish the field fertility tests [44], which is very laborious, expensive and time consuming [46].

Alternatively, embryo culture techniques allow exploring *in vitro* bull fertility. The employ‐ ment of such techniques has provided interesting but contradictory results regarding corre‐ lations between embryo *in vitro* embryo production (IVP) and *in vivo* bull fertility. Although positive correlations between IVP results and field fertility has been reported for some authors [12,14,16, 17, 20,46,48], other studies did not confirm the positive high correlations between *in vitro* fertilization (IVF) outcomes and *in vivo* fertility of evaluated sires [49,50,51]. However, Sudano et al*.* [12] recently demonstrated that it is possible to estimate bull fertility based on IVF outcomes, using a Bayesian statistical inference model.

Although interesting, it is still precipitated to ensure that the individual ability of fertilizing oocytes *in vitro* is a useful parameter for predicting *in vivo* bull fertility following AI. Hence, according to Ward et al. [20], a range of protocol variations among different IVP laboratories, the low repeatability in the results, as well as the various factors that may affect IVP outcomes, adds even more uncertainty if the *in vitro* ability for oocytes fertilization of a semen sample is sufficient accurate for predicting the sire field fertility. Additionally, it is noteworthy that more practical and/or simple laboratory techniques for assessing semen quality would be more advantageous for AI industry than the employment of IVP procedures.

polyunsaturated fatty acids [37]. Reactive oxygen species (ROS) may become cytotoxic through damage to proteins, nucleic acids and membrane lipids, if ROS concentrations overcome the natural defense mechanisms of the cell and extending medium [55]. Hence, since the high production of ROS might cause damages to plasma membrane structure, it can impair sperm function and motility [34,37]. A high degree of membrane lipid destabilization may lead to functional capacitation, reducing the sperm lifespan and fertilizing capacity [56]. In this sense, Hallap et al. [57] demonstrated that the amount of uncapacitated spermatozoa may provide

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Although the molecular basis involving the whole process of sperm capacitation has not yet been fully elucidated, it is recognized that sperm capacitation is a sequential event of bio‐ chemical alterations that involve numerous physiological changes. Some events related to the beginning of capacitation process include the removal of peripheral membrane factors, changes in membrane fluidity and in lipid composition [58,59]. Thus, the mammalian sperm capacitation is associated with reorganization of plasma membrane due to phospholipids redistribution of cholesterol removal [57]. Hence, the lipophilic probe Merocianina 540 may be used to monitor the level of phospholipid bilayer disorder of plasma membrane. Using this probe, the fluorescence intensity is increased with increasing membrane bilayer disorder, which can be an indicative of initial sperm capacitation process. In laboratory studies, this probe is commonly associated with the use of the probe Yo-Pro-1, which allows the simulta‐ neous analysis of plasma membrane integrity. This is due to the fact that Yo-Pro-1 is a specific DNA probe with excitation and emission of fluorescence similar to the Merocianina 540

As stated above, oxidative stress is a recognized contributor to defective sperm function [34,37,39,60]. Spermatozoa is very susceptible to peroxidative damage because of their high cellular content of polyunsaturated fatty acids that are particularly vulnerable to this form of stress [37]. Recently, a fluorescence assay using the fluorophore 4,4-di‐ fluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11- BODIPY581/591) has been successfully applied for detecting lipid peroxide formation in living bovine sperm cells [34]. This assay relies on the sensitivity of C11-BODIPY581/591, a fluorescent fatty acid conjugate, which readily incorporates into biological membranes [60]. Upon exposure to ROS, the C11-BODIPY581/591 responds to free radical attack with an irreversible shift in spectral emission from red to green that can be quantified by flow cy‐ tometry [37,60]. Still, it is noteworthy that the negative effect of some ROS-generating systems does not require lipid peroxidation to induce cytotoxic changes in spermatozoa. In this sense, Guthrie and Welch [61] observed that Menadione and H2O2 decreased the

In an interesting study, Kasimanickam et al. [39] reported that bull fertility was positively correlated to plasma membrane integrity and progressive motility. According to the authors, plasma membrane integrity significantly influenced the fertilizing capacity of a sire. Moreover, the authors demonstrated that plasma membrane integrity and progressive motility were negatively correlated to sperm lipid peroxidation and that lipid peroxidation and bull fertility was also high negatively correlated. Bulls with higher sperm lipid peroxidation were more

percentage of motile sperm but had no effect on BODIPY oxidation.

valuable information about frozen–thawed semen quality.

(around 540 nm) [57,58].

Correa et al.[11] observed that the total number of motile spermatozoa tended to be higher in high fertility bulls. Farrell et al. [41] demonstrated that multiple combinations of motility sperm variables obtained by Computer Assisted Semen Analysis (CASA) had higher correlations with bull field fertility than single parameters evaluated separately. The authors observed that the combination of Progressive Motility, ALH (amplitude of lateral sperm head displacement), BCF (sperm beat cross frequency), and VAP (Average Path Velocity) presented high correla‐ tion value (r2 = 0.87) and that the combination of ALH, BCF, linearity, VAP and VSL (Straight-Line Velocity) presented even higher correlation value (r2 = 0.98). Hence, it has been demonstrated that sperm motility evaluations are important for the assessment of semen quality, mainly when CASA is used for assessing semen motility patterns. This non-subjective sperm analysis provides an opportunity to assess multiple characteristics on a large sample of spermatozoa, which allows assessing several sperm motility parameters with high repeata‐ bility [33,41].

Even though that computer-based analysis provides high accuracy of *in vitro* motility evalu‐ ation [33,41], the assessment of different aspects related to sperm physiology may guarantee better investigation of semen quality [38,52]. Changes in membrane architecture and sperm compartment functionality may interfere with cellular competence and with the process of fertilization. These changes can be monitored using fluorescent probes that are able to bind and stain specific structures of the cell permitting a direct diagnosis [38]. Celeghini et al. [38,53] reported an efficient and high-repeatability technique for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function, using a combination of the following probes: propidium iodide (PI), fluorescein isothiocyanate–Pisum sativum agglutinin (FITC-PSA) and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide (JC-1) respectively.

Januskauskas et al. [35] found significant correlations between field fertility and plasma membrane integrity assessed by PI. Conversely, Brito et al. [54] reported no significant correlation between bovine *in vitro* fertilization (IVF) and plasma membrane integrity, measured by Eosin/Negrosin staining, CFDA/PI, SYBR-14/PI and HOST (hypo-osmotic swelling test). Nevertheless, Tartaglione and Ritta [36] demonstrated that the combination of plasma membrane integrity and functional laboratory tests presented high correlation coefficient with *in vitro* bull fertility. The authors demonstrated that combination of Eosin/ Negrosin staining test with HOST presented high correlation coefficient with *in vitro* fertility outcomes. When sperm plasma and acrosomal membrane integrity results (assessed by Trypam/Blue Giemsa staining) were included in the regression model, a higher correlation coefficient was obtained. The authors emphasized that higher is the capacity for predicting semen fertility when higher number of sperm evaluations is performed [36].

Another concern of semen fertility studies is the occurrence of sperm oxidative stress. Sper‐ matozoa are susceptible to oxidation of their plasma membranes due to the presence of polyunsaturated fatty acids [37]. Reactive oxygen species (ROS) may become cytotoxic through damage to proteins, nucleic acids and membrane lipids, if ROS concentrations overcome the natural defense mechanisms of the cell and extending medium [55]. Hence, since the high production of ROS might cause damages to plasma membrane structure, it can impair sperm function and motility [34,37]. A high degree of membrane lipid destabilization may lead to functional capacitation, reducing the sperm lifespan and fertilizing capacity [56]. In this sense, Hallap et al. [57] demonstrated that the amount of uncapacitated spermatozoa may provide valuable information about frozen–thawed semen quality.

adds even more uncertainty if the *in vitro* ability for oocytes fertilization of a semen sample is sufficient accurate for predicting the sire field fertility. Additionally, it is noteworthy that more practical and/or simple laboratory techniques for assessing semen quality would be more

Correa et al.[11] observed that the total number of motile spermatozoa tended to be higher in high fertility bulls. Farrell et al. [41] demonstrated that multiple combinations of motility sperm variables obtained by Computer Assisted Semen Analysis (CASA) had higher correlations with bull field fertility than single parameters evaluated separately. The authors observed that the combination of Progressive Motility, ALH (amplitude of lateral sperm head displacement), BCF (sperm beat cross frequency), and VAP (Average Path Velocity) presented high correla‐ tion value (r2 = 0.87) and that the combination of ALH, BCF, linearity, VAP and VSL (Straight-

demonstrated that sperm motility evaluations are important for the assessment of semen quality, mainly when CASA is used for assessing semen motility patterns. This non-subjective sperm analysis provides an opportunity to assess multiple characteristics on a large sample of spermatozoa, which allows assessing several sperm motility parameters with high repeata‐

Even though that computer-based analysis provides high accuracy of *in vitro* motility evalu‐ ation [33,41], the assessment of different aspects related to sperm physiology may guarantee better investigation of semen quality [38,52]. Changes in membrane architecture and sperm compartment functionality may interfere with cellular competence and with the process of fertilization. These changes can be monitored using fluorescent probes that are able to bind and stain specific structures of the cell permitting a direct diagnosis [38]. Celeghini et al. [38,53] reported an efficient and high-repeatability technique for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function, using a combination of the following probes: propidium iodide (PI), fluorescein isothiocyanate–Pisum sativum agglutinin (FITC-PSA) and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide

Januskauskas et al. [35] found significant correlations between field fertility and plasma membrane integrity assessed by PI. Conversely, Brito et al. [54] reported no significant correlation between bovine *in vitro* fertilization (IVF) and plasma membrane integrity, measured by Eosin/Negrosin staining, CFDA/PI, SYBR-14/PI and HOST (hypo-osmotic swelling test). Nevertheless, Tartaglione and Ritta [36] demonstrated that the combination of plasma membrane integrity and functional laboratory tests presented high correlation coefficient with *in vitro* bull fertility. The authors demonstrated that combination of Eosin/ Negrosin staining test with HOST presented high correlation coefficient with *in vitro* fertility outcomes. When sperm plasma and acrosomal membrane integrity results (assessed by Trypam/Blue Giemsa staining) were included in the regression model, a higher correlation coefficient was obtained. The authors emphasized that higher is the capacity for predicting

Another concern of semen fertility studies is the occurrence of sperm oxidative stress. Sper‐ matozoa are susceptible to oxidation of their plasma membranes due to the presence of

semen fertility when higher number of sperm evaluations is performed [36].

= 0.98). Hence, it has been

advantageous for AI industry than the employment of IVP procedures.

6 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

Line Velocity) presented even higher correlation value (r2

bility [33,41].

(JC-1) respectively.

Although the molecular basis involving the whole process of sperm capacitation has not yet been fully elucidated, it is recognized that sperm capacitation is a sequential event of bio‐ chemical alterations that involve numerous physiological changes. Some events related to the beginning of capacitation process include the removal of peripheral membrane factors, changes in membrane fluidity and in lipid composition [58,59]. Thus, the mammalian sperm capacitation is associated with reorganization of plasma membrane due to phospholipids redistribution of cholesterol removal [57]. Hence, the lipophilic probe Merocianina 540 may be used to monitor the level of phospholipid bilayer disorder of plasma membrane. Using this probe, the fluorescence intensity is increased with increasing membrane bilayer disorder, which can be an indicative of initial sperm capacitation process. In laboratory studies, this probe is commonly associated with the use of the probe Yo-Pro-1, which allows the simulta‐ neous analysis of plasma membrane integrity. This is due to the fact that Yo-Pro-1 is a specific DNA probe with excitation and emission of fluorescence similar to the Merocianina 540 (around 540 nm) [57,58].

As stated above, oxidative stress is a recognized contributor to defective sperm function [34,37,39,60]. Spermatozoa is very susceptible to peroxidative damage because of their high cellular content of polyunsaturated fatty acids that are particularly vulnerable to this form of stress [37]. Recently, a fluorescence assay using the fluorophore 4,4-di‐ fluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11- BODIPY581/591) has been successfully applied for detecting lipid peroxide formation in living bovine sperm cells [34]. This assay relies on the sensitivity of C11-BODIPY581/591, a fluorescent fatty acid conjugate, which readily incorporates into biological membranes [60]. Upon exposure to ROS, the C11-BODIPY581/591 responds to free radical attack with an irreversible shift in spectral emission from red to green that can be quantified by flow cy‐ tometry [37,60]. Still, it is noteworthy that the negative effect of some ROS-generating systems does not require lipid peroxidation to induce cytotoxic changes in spermatozoa. In this sense, Guthrie and Welch [61] observed that Menadione and H2O2 decreased the percentage of motile sperm but had no effect on BODIPY oxidation.

In an interesting study, Kasimanickam et al. [39] reported that bull fertility was positively correlated to plasma membrane integrity and progressive motility. According to the authors, plasma membrane integrity significantly influenced the fertilizing capacity of a sire. Moreover, the authors demonstrated that plasma membrane integrity and progressive motility were negatively correlated to sperm lipid peroxidation and that lipid peroxidation and bull fertility was also high negatively correlated. Bulls with higher sperm lipid peroxidation were more likely to have a high DNA fragmentation and low plasma membrane integrity. Also, these bulls presented lower chances of siring calves [39]. These results are in accordance with Zabludovsky et al. [62] which also had demonstrated negative correlations between lipid peroxidation and IVF fertilization outcomes in humans.

An interesting study of [32] demonstrated that the average of sperm head shape identified to be from high fertility bulls was more tapered and elongated (more elliptical) than the average shape of sperm identified to be from low fertility bulls. In addition, the authors observed that quantifying changes in sperm shape can be detected by Fourier parameters, which characterize the curvilinear perimeter of sperm head using harmonic amplitudes to describe the sperm nuclear shape. The relationship between sire fertility and Fourier parameters of sperm morphometric analysis was investigated. It was observed that Fourier descriptors were able to detect small differences in sperm nuclear shape from bulls with different fertility [32;64]. According to [63], the most promising method of quantifying changes in sperm head shape is

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9

Acevedo et al. [70] reported that spermatogenic disturbance resulted in production of abnor‐ mal sperm and that sperm DNA vulnerability to acid denaturation was positively associated with sperm having misshapen heads. This provided more support for the assertion that occurrence of sperm with misshapen heads can signal chromatin abnormalities and potential incompetence for fertilization of a semen sample [63]. Kasimanickam et al. [39] reported that some deleterious effects of sperm lipid peroxidation are also related to impairment in sperm DNA, which may also reduce bull fertilizing potential. The sires with high sperm DNA fragmentation index presented lower sperm fertilization potential; whereas sires with lower

Besides the intense efforts from worldwide researchers, until now, no single laboratory test has accurately predicted the real fertilizing capacity of a semen sample [52, 71]. Hence, in spite of some interesting results of *in vitro* sperm characteristics, a notable consideration is the importance of field trials when definitive conclusions are taken regarding semen fertility.

Individual bulls may differ in their ability to fertilize oocytes and/or to develop to blastocyst stages after *in vitro* and *in vivo* fertilization procedures. Hence, the success of bovine repro‐ ductive programs largely depends on the use of good quality semen. When only high fertility bulls are used, better fertilization rates and reproductives outcomes are achieved, increasing

The sequence of insemination after simultaneous thawing of multiple semen straws may present different effect and/or relevance on fertility outcomes, depending on the sire that is being used in the reproductive program. However, the reason why semen from some bulls seems to be more susceptible and/or differently affected to specific procedures, semen handling protocols, and/or environments remains to be further investigated. It is noteworthy, though, that the use of different sires, semen extenders, thawing bath volumes, semen straw volumes, AI technicians, semen handling procedures, number of AI guns utilized, ambient conditions, farm management and cow categories, as well as the use of different laboratory

DNA fragmentation index presented higher chance of siring calves [39].

the reproductive efficiency and thus, reducing the costs of the programs.

analyses, might generally influence the results obtained.

utilizing the Fourier harmonic amplitude analysis.

**4. Conclusions and implications**

It has frequently been reported that low-fertility bulls generally had high seminal content of morphologically abnormal cells [63]. Sperm with classically misshapen heads did not access the egg following AI since they do not traverse the female reproductive tract and/or participate in fertilization [43]. Some geometrical alterations of head morphology can cause differences in sperm hydrodynamics. According to [63], abnormal-shaped heads should be of primary concern regarding male fertility. The recognition of uncompensable cells in the ejaculate is currently best based on abnormal levels of sperm with misshapen heads [63].

Ostermeier et al. [32,64] also observed that some sperm morphometric variables were able to detect small differences in sperm nuclear shape which seems to be related to sire fertility. According to Beletti et al. [65], the application of computational image analysis for morpho‐ logical characterization allows the identification of minor morphometric alterations of sperm head. However, little is known about the influence of such abnormalities on bull fertility. Because mammalian sperm heads consist almost entirely of chromatin, even minor changes in chromatin organization might affect sperm head shape. Nonetheless, morphological alterations in sperm head are not always caused by alterations in chromatin condensation. In the same way, chromatin abnormalities are not always followed by evident morphological irregularities [32,65,66].

A number of methods are available for identifying alterations in the stability of sperm chromatin. Sperm chromatin structure analysis (SCSA), currently the most used of these methods, is based on a flow cytometric evaluation of the fluorescence of spermatozoa stained with acridine orange [32,67]. Another method for chromatin evaluation uses a cationic dye, toluidine blue (at pH 4.0) that exhibits metachromasy. This dye binds to ionized phosphates in the DNA. In normal sperm chromatin, few dye molecules bind to DNA; this result in staining that varies from green to light blue. Spermatozoa with less compacted chromatin have more binding sites for the dye molecules, resulting in staining that varies from dark blue to magenta [65].

Whereas human-based methods for assessing sperm parameters involve a high degree of subjectivity in the visual analysis, computer-based methods for image processing and analysis are currently available. It can provide a more objective evaluation of cell motility and sperm morphological abnormalities, in addition to greater sensitivity, accuracy, speed and reprodu‐ cibility. Computational morphometric analysis of spermatozoa usually considers basic measurements like the area, perimeter, length and width, as well as features derived from the measurements, such as the width:length ratio, shape factor and others [68]. An interesting approach is to use image analysis to characterize the sperm chromatin in smears stained with toluidine blue which also allows a morphometric analysis to be done concomitantly with the investigation of chromatin [65,69].

An interesting study of [32] demonstrated that the average of sperm head shape identified to be from high fertility bulls was more tapered and elongated (more elliptical) than the average shape of sperm identified to be from low fertility bulls. In addition, the authors observed that quantifying changes in sperm shape can be detected by Fourier parameters, which characterize the curvilinear perimeter of sperm head using harmonic amplitudes to describe the sperm nuclear shape. The relationship between sire fertility and Fourier parameters of sperm morphometric analysis was investigated. It was observed that Fourier descriptors were able to detect small differences in sperm nuclear shape from bulls with different fertility [32;64]. According to [63], the most promising method of quantifying changes in sperm head shape is utilizing the Fourier harmonic amplitude analysis.

Acevedo et al. [70] reported that spermatogenic disturbance resulted in production of abnor‐ mal sperm and that sperm DNA vulnerability to acid denaturation was positively associated with sperm having misshapen heads. This provided more support for the assertion that occurrence of sperm with misshapen heads can signal chromatin abnormalities and potential incompetence for fertilization of a semen sample [63]. Kasimanickam et al. [39] reported that some deleterious effects of sperm lipid peroxidation are also related to impairment in sperm DNA, which may also reduce bull fertilizing potential. The sires with high sperm DNA fragmentation index presented lower sperm fertilization potential; whereas sires with lower DNA fragmentation index presented higher chance of siring calves [39].

Besides the intense efforts from worldwide researchers, until now, no single laboratory test has accurately predicted the real fertilizing capacity of a semen sample [52, 71]. Hence, in spite of some interesting results of *in vitro* sperm characteristics, a notable consideration is the importance of field trials when definitive conclusions are taken regarding semen fertility.
