Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=−0.789, P<0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Δψm) post-thaw (r=−0.689, P<0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, P<0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering ‘apoptotic-like changes’ that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

The accurate prediction of bull fertility is of major economic importance in the dairy breeding industry. Sperm fertilizing potential is determined by their ability to reach the oocyte, complete fertilization, and sustain embryogenesis, which is partly determined by the quality of sperm DNA. In the present study, we analyzed several sperm functions required for fertility, including DNA damage, in frozen-thawed spermatozoa of breeding bulls with different adjusted nonreturn rates (NRR56), and identified a suitable combination of parameters that could be used to predict bull fertility. Based on the NRR56, bulls were classified into below- and above-average fertility, a total of 37 characteristics of spermatozoa were evaluated for each bull, and their relationship with bull fertility was studied. Of the different sperm functional attributes, differences were observed in sperm viability, acrosomal integrity, reactive oxygen species, and DNA fragmentation index (%DFI) among below-average, average, and above-average fertility bulls. Principal component analysis also revealed that sperm viability, acrosome status, reactive oxygen species, and %DFI were the important variables, having highest correlation with NRR56. Our results indicated that the proportion of live [correlation coefficient (r) = 0.53] and live acrosome-reacted spermatozoa (r = 0.50) were significantly positively related to NRR56, whereas the proportion of dead spermatozoa (r = -0.53) and %DFI (r = 0.61) were significantly negatively related to NRR56 in bulls. Linear regression analysis indicated that a combination of live [coefficient of determination (R2) = 0.72], dead (R2 = 0.72), live hydrogen peroxide-negative spermatozoa (R2 = 0.64), and %DFI (R2 = 0.56) could differentiate below-average and above-average fertility bulls, and thus were considered for development of a fertility prediction model. The accuracy of the developed model for fertility prediction in bulls was high (R2 = 0.83). We concluded that flow cytometric detection of sperm viability, hydrogen peroxide status, and %DFI could discriminate below- from above-average fertility bulls.

Since bull fertility may be adversely affected by hot humid conditions, the current increase in global temperature is of concern for future livestock production. Heat stress occurs when the body's normal physiological mechanisms to regulate body temperature cannot cope with external conditions. The testes and scrotum have their own complex regulatory mechanisms to protect developing sperm during their most vulnerable stages, but even these may be overwhelmed by unfavourable external conditions. The effects of mild, moderate and severe heat stress are somewhat different, with cattle exposed to mild and moderate heat stress apparently showing an adverse effect on fertility, whereas cattle in very hot, humid climates almost continuously may not exhibit any difference in sperm quality throughout the year. This apparent paradox may be due to differences in the cattle populations being studied, since they could differ in breed, age, purpose (beef versus dairy), or even in the methods used to assess sperm quality. The adverse effects on fertility may occur through the effects of reactive oxygen species on sperm DNA, or through perturbation of the production of antioxidants that usually protect sperm from oxidative attack. These effects can be mitigated to some extent by choosing breed and age of bulls with care, and adopting breeding strategies that avoid semen collection or ejaculation at the most adverse times of year. Husbandry measures such as controlled ventilation, misting, provision of shade or cool surfaces for lying down, could aid temperature regulation. Avoiding heat stress during late pregnancy aids calf growth in early life; careful feeding regimens for young bull calves create good conditions for sperm quality after puberty. Bull fertility is too important to be left to chance. Breeds should be chosen according to climate conditions and the purpose of livestock production.

The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.

Antibiotics are added to semen extenders to be used for artificial insemination (AI) in livestock breeding to control bacterial contamination in semen arising during collection and processing. The antibiotics to be added and their concentrations for semen for international trade are specified by government directives. Since the animal production industry uses large quantities of semen for artificial insemination, large amounts of antibiotics are currently used in semen extenders. Possible alternatives to antibiotics are discussed, including physical removal of the bacteria during semen processing, as well as the development of novel antimicrobials. Colloid centrifugation, particularly Single Layer Centrifugation, when carried out with a strict aseptic technique, offers a feasible method for reducing bacterial contamination in semen and is a practical method for semen processing laboratories to adopt. However, none of these alternatives to antibiotics should replace strict attention to hygiene during semen collection and handling.

Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to investigate the possibility of using these tests predictively.