**4. Conclusions and implications**

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

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

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

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

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

currently best based on abnormal levels of sperm with misshapen heads [63].

peroxidation and IVF fertilization outcomes in humans.

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

irregularities [32,65,66].

that varies from dark blue to magenta [65].

investigation of chromatin [65,69].

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

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 analyses, might generally influence the results obtained.

Worth mentioning though, that when the correct semen handling recommendation is provid‐ ed, as well as the adequate cautious and/or proficiency of AI technician is assured, the sequence of insemination is not likely to severely impact semen quality and reproductive performance in AI programs. Thus, it is deemed reasonable to attempt to the fact that the care and concern with semen storage and handling is essential to obtain satisfactory reproductive outcomes after AI. In addition, greater attention should be directed to the simultaneous thawing of multiple semen straws, especially when the thawing procedures do not include a thermostatically controlled water-bath unit.

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Even though that an *in vitro* semen assay for determining bull fertility would be of great benefit to AI programs, it is unlikely that the evaluation of a single sperm characteristic may reflect the real sperm fertilization capacity of a semen sample, considering the complexity of the reproductive process.

In spite of the promising results reported above, until now, no single laboratory test was able to accurately predict, with the required repeatability, the real fertilizing capacity of a sire. Hence, potential bull fertility can be estimated from laboratory semen assessment with higher accuracy when a combination of several *in vitro* sperm analysis is performed.

Still, further studies contributing to the understanding of seminal differences among bulls that might be related to differences in fertility rates commonly observed in AI programs must be encouraged.
