Cooled storage of semen from livestock animals (part I): boar, bull, and stallion.

This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques for cooled storage of semen from livestock animals. The first part describes the current state of the art of liquid semen preservation in boars, bulls, and stallions, including the diluents, use of additives, processing, temperature, and cooling of semen. The species-specific physiology and varying extents of cold shock sensitivity are taken into consideration. In addition, factors influencing the quality of cooled-stored semen are discussed. Methods, trends, and the most recent advances for improving sperm quality during cold-temperature storage are highlighted and their respective advantages and disadvantages are contrasted. There has been much progress in recent years regarding cold-temperature storage of boar sperm and there is great potential for a large-scale use to replace the current 17 °C temperature storage regime and the associated use of antibiotics in the future. For stallion sperm, there is an opposite trend away from previous low-temperature storage towards storage at higher temperatures to increase sperm viability and longevity. In bulls, liquid storage of sperm is mostly used in the seasonal dairy production systems of New Zealand and Ireland, but with further research focusing on shelf-live elongation of liquid preserved sperm, there is potential for an application in breeding programs worldwide.

Antibiotics and their alternatives in Artificial Breeding in livestock.

Antibiotics are mandatory components of semen extenders for the control of bacterial contamination and growth. The increasing rate of worldwide resistance to conventional antibiotics in semen preservation media requires the development of new antimicrobial alternatives. This review provides an update on this topic and also highlights the improvement of hygiene in Artificial Insemination centers in order to prevent the development of bacterial resistance. Ideas are shared on future diagnostic tools for bacterial contamination in Artificial Breeding. Finally, new methods to remove or reduce bacteria in semen will be discussed.

Impact of hygiene on bacterial contamination in extended boar semen: An eight-year retrospective study of 28 European AI centers.

Antibiotic agents such as gentamicin represent essential components of semen extenders in order to reduce bacterial contamination. But antibiotic resistance increases and AI centers start utilizing antibiotic agents which are more potent. Therefore, a shift to preventing bacterial contamination has to take place. In this study, we could demonstrate that hygiene is a tool capable of reducing bacterial load. In order to analyze 1434 extended semen samples and nine specially established hygienic critical control points (HCCPs, n = 828), 92 quality control audits have been carried out in a time period from 2012 until 2019 in 28 European AI centers. The results show the process of introducing a basic hygienic standard in audit 1 (2012/2013) and 2 (2014/2015) and the resulting achievements by means of improved hygienic conditions in audit 3 (2016/2017) and 4 (2018/2019). Within the scope of audit 1, 19% of the semen samples were contaminated with bacteria (cutoff ≥100 colony-forming units/mL). Audit 2 showed a bacterial load of 13.6% whereas during audit 3 and 4 very low bacterial contamination rates were recorded (4.5 and 5.5%, respectively). In the same manner, analysis of hygiene at different CCPs during semen production showed a decrease in all average HCCP-scores (score 1-6) comparing audit 4 to 1. By regression analysis we could show a significant audit-dependent association of the bacterial contamination in semen samples and hygiene of HCCPs. Furthermore, analysis of the odds ratio (OR) reveals that the bacterial contamination of certain HCCPs poses an increased risk of receiving bacterially contaminated semen samples (filling machine: OR = 3.02, P = 0.06; extender: OR = 8.97, P < 0.001; inner face of dilution tank lids: OR = 3.14, P = 0.09). Around 60% of the variance of the bacterial contamination in semen samples could be explained by hygienic conditions at different control points and their interaction with audit period and AI center. Antimicrobial agents are essential to protect human and animal health but excessive or inappropriate use can lead to the emergence of resistant bacteria. As shown in our study, hygiene management can significantly reduce bacterial contamination and is therefore capable of preventing antibiotic resistance.

New trends in production management in European pig AI centers.

Reducing the number of spermatozoa per artificial insemination (AI) dose and managing semen in ways to ensure greater quality at the same time represents current challenges with sperm processing in pig AI centers. Based on a multi-year comparative analysis of process steps in different pig AI centers, and complementary experimental studies under standardized laboratory conditions, current process standards for the preservation of boar semen have been updated and new ones developed. Currently, these standards represent an integral part of the quality assurance of 29 European pig AI centers in ten different organizations in Germany, Switzerland and Austria. Improvement of hygiene management and guidelines for prudent use of antibiotics have become key issues. Furthermore, new quality control tools have been implemented in the processing and transport of boar semen: e.g. refractometry as an easy-to-use tool to estimate extender osmolarity and 'mobile sensing' apps for continuous monitoring of various environmental parameters. Moreover, based on a series of experiments under laboratory and field conditions, guidelines for optimizing the dilution process, and time and temperature management during boar semen processing, have been developed and implemented. Similarly, recommendations for the handling of semen doses during storage have been renewed. Over the years, the efficiency of the quality assurance system has been reflected by a decrease of bacterial contamination and a concomitant increase in the quality of semen doses. In conclusion, science-based quality assurance is an effective way to improve the production performance in pig AI centers, resulting in high quality and economically-priced semen for pig producers. Increasing knowledge of sperm physiology together with computational and technical innovations will continue to develop and modify quality assurance concepts in the future.

Investigations on the endometrial response to intrauterine administration of N-acetylcysteine in oestrous mares.

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.

Effects of oral treatment with N-acetylcysteine on the viscosity of intrauterine mucus and endometrial function in estrous mares.

Persistent breeding-induced endometritis is ranked as the third most common medical problem in the adult mare and leads to enormous economic loss in horse breeding. In mares suffering from persistent breeding-induced endometritis, increased amounts of intrauterine (i.u.) fluid or viscous mucus in estrus or after breeding may act as a barrier for sperm and can contribute to low fertility. Current therapies of these mares aim to eliminate i.u. fluid and mucus by uterine lavage and/or administration of ecbolic drugs. Recently, i.u. administration of N-acetylcysteine (NAC) has been shown to support therapy in mares with endometritis. It was the objective of the present study to investigate effects of an oral administration of NAC on the viscosity of i.u. fluid in estrous mares. It was hypothesized that oral treatment with NAC reduces the viscosity of i.u. fluid and has a positive effect on the inflammatory response of the endometrium. Mares (n = 12) were included in the study as soon as estrus was detected (ovarian follicle >3.0 cm and endometrial edema), which was defined as Day 1. They were randomly assigned to a treatment (10 mg/kg NAC on Days 1-4) or a control group (no treatment). On days 1 and 5 i.u. mucus was collected and its rheologic properties were accessed. On Day 5, endometrial biopsies were obtained and evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid Schiff (PAS). In the treatment group, viscosity of i.u. mucus increased significantly between Days 1 and 5 (P < 0.05), while no differences were found in control mares (n.s.). At no time were significant differences between treated and control mares seen. Integrity of epithelium was not affected. After NAC treatment the mean number of PMN in endometrial biopsies was significantly lower compared to mares of the control group (1.9 ± 0.3 vs. 4.8 ± 0.4; P < 0.05). Nuclear immunostaining for COX2 was significantly lower after NAC treatment compared to control mares (P < 0.05). Score for PAS and Alcain staining of mucus in deep uterine glands differed significantly between groups (both P < 0.05). We conclude that oral NAC treatment does not reduce viscosity of uterine mucus but has an antiinflammatory effect on the equine endometrium.