Suitability of semen stress tests for predicting fertilizing capacity of boar ejaculates.

Besides classical semen parameters, semen stress tests (SSTs) are helpful tools to assess the fertilizing capacity of sperm. However, valid studies on SSTs in relation to fertility are rare because several corrections of common fertility parameters for female and male effects are required. Therefore, over a one-year period, we analyzed semen parameters of 260 ejaculates obtained from 130 Pietrain boars aged between 8 and 9 months in one AI center as well as 1521 corresponding insemination records for these ejaculates. Two consecutive ejaculates (4th and 5th) were collected from each boar and extended in DiluPorc™ BTS. In addition to routine semen evaluation, sperm motility was assessed after heat-resistance test (300 min incubation at 38 °C after seven days storage at 16 °C, HRT) and cold-resistance test (10 min incubation at 38 °C after three days storage at 6 °C, CRT). Generalized linear models (GLMs) were applied to analyze effects for the following predictors of farrowing rate (FR), number of total born (NTB) and live born (NLB) piglets: farm (P = 0.013 [FR], P = 0.001 [NTB], P = 0.023 [NLB]), parity (P = 0.679, P = 0.01, P < 0.001), weekday (P = 0.012, P = 0.08, P = 0.009) and year × season (P < 0.001, P = 0.688, P = 0.574). On boar level, GLMs revealed significant effects on FR, NTB and NLB for the predictors sow (all P < 0.001), total sperm number per dose (P = 0.007, P = 0.002, P < 0.001), total sperm motility (P = 0.002, P = 0.2, P = 0.003) and mitochondrial activity (P = 0.004, P < 0.001, P = 0.002). Moreover, FR and NTB were influenced by membrane integrity (both P < 0.001), FR and NLB by cold-resistance (P < 0.001, P = 0.043), and NTB and NLB by sperm morphology (P = 0.001, P < 0.001) and boar (both P < 0.001). NLB was additionally influenced by heat-resistance (P = 0.004) and farm (P = 0.018) and solely NTB was influenced by sperm output (P = 0.03). Boar and semen related factors explained 9% of the total variation in NTB and 7% of the total variation in NLB. Only 14.2% (n = 37) of the samples were both cold- and heat-resistant (≥65% of motile sperm). Cold- and heat-resistance were dependent factors (Chi-square, P = 0.001) and sperm motility after CRT and HRT showed a moderate positive correlation (rs = 0.40, P < 0.001, Spearman's rho). Finally, ROC curves demonstrated that neither SST can be used as a sole test for predicting the fertilizing capacity of boar ejaculates.

Development of a new antimicrobial concept for boar semen preservation based on bacteriocins.

The conventional storage temperature of 16-18 °C provides optimal conditions for the preservation of boar sperm quality, which are extremely cold sensitive cells. On the other hand, however, it requires the addition of antibiotics to inhibit bacterial growth. Rising numbers of antibiotic resistant bacteria call for alternatives to this conventional storing method. As potential alternative, three different bacteriocin candidates with known bacteriolytic activity against E. coli were examined on possible negative effects concerning the sperm quality and on their impact on bacterial growth of E. coli ILSH 02692 in BTS-extended semen w/o antibiotics. Although the lower concentrations (0.01 and 0.25%) of all bacteriocins did not show any impact on the quality of the semen, the higher concentrations (0.5 and 1.0%) of two bacteriocins led to a significant (P < 0.05) reduction in several sperm quality characteristics. The bacteriocin 860/1c after AMS/dialysis did not affect the sperm quality in any of the tested concentrations and in all tested extenders (BTS, MIII, Androstar Premium and Androhep all w/o antibiotics) at 16 °C as well as at 6 °C. This bacteriocin reduced growth of E. coli ILSH 02692 in BTS-extended semen by 50% compared to the control w/o bacteriocin. Furthermore, a preliminary insemination trial indicated no impact of the selected bacteriocin on fertility. These promising results show that the application of bacteriocins in liquid-preserved semen is a feasible possibility in the future.

Evaluation of a panel of spermatological methods for assessing reprotoxic compounds in multilayer semen plastic bags.

The increase of fertility performance in sows is one of the biggest achievements in pig production over the last 30 years. Nevertheless, pig farms using artificial insemination (AI) repeatedly experienced in recent year's fertility problems with dramatic consequences due to toxic compounds from plastic semen bags. In particular, bisphenol A diglycidyl-ether (BADGE) present in multilayer plastic bags can leach into the semen and could affect the functionality of the spermatozoa. Former studies could not find any alterations in spermatozoa based on the exposure to BADGE. The aim of the study was to evaluate effects of BADGE on boar spermatozoa using an extended panel of spermatological methods. In spring 2019, a large drop in farrowing rates from 92.6 ± 2.3% to 63.7 ± 11.1% in four sow farms in Croatia was detected. In migration studies, BADGE could be identified as a causal toxic compound and leached into the extended semen in concentration of 0.37 ± 0.05 mg/L. Detailed spermatological studies showed that significant predictors for effects on spermatozoa were different levels of motility and kinematic data after a prolonged storage time, thermo-resistance test (prolonged incubation time), mitochondrial activity, membrane integrity and fluidity. No serious effects were observed for sperm morphology and DNA fragmentation. These results provide new insights into the development of a new quality assurance concept for a detailed spermatological examination during testing of plastic materials for boar semen preservation. It could be shown that boar spermatozoa are an excellent biosensor to detect potential toxicity and fertility-relevant compounds.

Antibacterial defense in bull and boar semen: A putative link to the microbiome and reproductive strategy?

Several domestic and wildlife species have been shown to possess antibacterial defenses in their ejaculate most probably in order to increase the fertilization success and protect against sexually transmitted pathogens. However, very little is known about the consequences and factors influencing the differences within and between species as far as ejaculate-associated immunity. In the present study, we have analyzed bacterial killing activity (BKA) against Escherichia (E.) coli and Staphylococcus (S.) aureus as well as lysozyme concentrations (LC) in seminal plasma from 60 Fleckvieh bulls. Further, sperm quality and its association with BKA and LC were determined. Twenty percent of the individuals displayed BKA against both bacteria, 78.3% against S. aureus only and 1.7% of the bulls did not indicate any BKA in seminal plasma. No bulls with seminal plasma BKA only against E. coli were identified; implying that 80.0% of the tested bulls had no ejaculate associated defense mechanisms against this gram-negative bacterial species in place. This is in striking contrast to results of Pietrain boars within our previous study, in which 42.8% of the 119 boars expressed an antibacterial activity against E. coli in seminal plasma, 10.9% amongst them with BKA against E. coli only. LC was higher in the bull group with BKA against both bacteria (1.2 ± 0.6 µg/mL) compared to the group with BKA against S. aureus only (0.7 ± 0.3 µg/mL), but - if calculated over all individuals - LC in bulls (0.8 ± 0.4 µg/mL) was lower compared to boars (2.4 ± 1.2 µg/mL). LC showed positive correlations to the age of the bulls and sperm quality as well as a negative relation to bacterial load in raw semen although the highest bacterial contamination was found in animals with seminal plasma BKA against both strains. We discuss the obtained results with regards to possible differences within the microbiome of female and male genital tracts and the reproductive strategies (vaginal vs. uterine depositors) in these two livestock species. Besides identifying the responsible molecules, future phylogenetically controlled comparative studies are needed for a better understanding of the evolution of species differences in ejaculate-associated antibacterial defenses.

Low temperature preservation of porcine semen: influence of short antimicrobial lipopeptides on sperm quality and bacterial load.

Antimicrobial resistance is a steadily increasing problem and poses a serious threat to global public health. Therefore, it is highly necessary to advance the development of novel antimicrobial compounds and semen preservation strategies. The aim of this study was to evaluate a low temperature, antibiotic-free preservation procedure using Androstar Premium (ASP) extender (Minitüb) with antimicrobial lipopeptides. Firstly, seven lipopeptides in two concentrations (1 × minimum inhibitory concentration (MIC)/2 × MIC) were tested on their sperm-compatibility at 17 °C. Two lipopeptides, C16-KKK-NH2 and C16-KKKK-NH2, did not negatively affect sperm quality and were further evaluated for their efficiency of bacterial growth inhibition at 5 °C. Besides an overall diminution of colony forming units, both peptides showed a reduction of bacterial subcultures (n = 103) with a decrement in Gram-positive rods from 65 (ASP w/o supplements) to 39/52 (ASP w/ C16-KKK-NH2/C16-KKKK-NH2), in Gram-positive cocci from 21 to 9/10 and in Gram-negative species from 17 to 8/5 total subcultures. Furthermore, lipopeptides revealed activity towards selected bacteria of potential concern in artificial insemination like Trueperella pyogenes, Alcaligenes faecalis, Pseudomonas aeruginosa (not C16-KKK-NH2), Pasteurella sp., Providencia stuartii, Escherichia coli (not C16-KKKK-NH2) and Streptococcus porcinus (not C16-KKKK-NH2). Consequently, both tested lipopeptides are promising candidates for alternative antibiotic-free preservation techniques of boar semen.

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.

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.

Development of predictive models for boar semen quality.

In this study a prognosis model is developed that predicts sperm quality characteristics based on external factors such as barn climate conditions, seasonality, semen collection frequency, age and breed of artificial insemination (AI) boars. For this a k-fold cross validation framework is used to test the prediction accuracy of a wide range of regression models that are based on different functional forms (linear, log-linear) and estimation techniques (ordinary least squares, seemingly unrelated regression, two-stage least squares estimation and three-stage least squares estimation). The dataset includes 241 boars from three barns within one boar stud located in Southern Germany, consisting of 7455 ejaculates collected during one year. The winner model predicts sperm motility with little error (Mean Absolute Percentage Error (MAPE): 4.35%), but is of limited use to predict sperm output (MAPE: 23.92%) and especially morphologically abnormal spermatozoa (MAPE: 44.67%). An estimation of marginal effects shows, that once confounding variables are controlled for, the considered barn climate variables do not have a measurable effect on sperm quality. Other factors have a more significant effect on sperm quality, like morphology-motility linkages, sperm concentration, interval between semen collections and to a lesser extent age and breed of the AI boar.

In vitro supplementation with unsaturated fatty acids improves boar sperm viability after storage at 6 °C.

Liquid preservation of the cold-sensitive boar sperm at a lesser temperature than the standard 17 °C would reduce bacterial growth and minimize the use of antibiotics. There was assessment, therefore, of the capacity of individual fatty acids bound to fatty acid free BSA to improve sperm survival at 6 °C because oxidative stress and lipid degradation are prominent detrimental factors. Different effects of the fatty acids were observed. Supplementation with naturally occurring fatty acids (linolenic, linoleic, oleic, palmitoleic acid), which may become metabolically incorporated into sperm lipids, increased the number of motile and progressively motile sperm after 2 days of storage during a thermo-resistance test (5 h at 38 °C) to that of control samples preserved at 17 °C in pure Beltsville Thawing Solution. With the exception of linolenic acid, all naturally occurring fatty acids enhanced the number of sperm with active mitochondria after 3 days of storage. Palmitoleic acid was the most effective supplement with effects already present when sperm were re-warmed for 30 min after 2 and 7 days of storage. The non-endogenous, non-integrated timnodonic acid (20:5) had no effect on sperm variables. Because the application of individual fatty acids attached to BSA had differing effects in preserving boar sperm at 6 °C, the use of combinations of fatty acids could be more efficacious than with use of natural lipid supplements for low temperature preservation of sperm.

Influences on thermo-resistance of boar spermatozoa.

The aim of this study was to investigate the influence of semen production management in 25 European boar studs on thermo-resistance (TRT) of boar spermatozoa. TRT is an important semen characteristic, easy to determine, and is used to explain variations in pig fertility. During a study period from 2013 to 2018, 905 ejaculates were analyzed for TRT on day 7 of semen storage. Statistical models showed that around 40% of the total variability in TRT could be explained by production management factors. Significant selected predictors were year, month, boar stud, boar age, dilution steps, mitochondrial activity and membrane integrity of spermatozoa, bacterial contamination, arrival temperature of ejaculates in the analysis laboratory, sperm concentration, ejaculate volume, sperm output, dose/ejaculate, and electrical conductivity of pure water for extender preparation. TRT increased during the study period. No effects were observed for breed, dilution rate, morphologically intact spermatozoa, and refractive index of extender on TRT. A holistic view of the requirements in the field of boar semen production is of great importance for future developments of individualized Hazard Analysis and Critical Control Point concepts, prognosis models for boar semen quality, and could help to improve the efficiency of AI organizations.

Semen cryopreservation and radical reduction capacity of seminal fluid in captive African lion (Panthera leo).

Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 µL containing 20 × 106 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 106 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.

Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry.

After leaving the testis, spermatozoa undergo several important steps of biochemical maturation during the passage through the epididymis, increasing their motility and fertilizing ability. These changes comprise (among others) the modification of the phospholipid composition of the sperm membrane. This process is thought to be important for the achievement of motility and fertilizing capacity. The lipids of the sperm membrane are characterized by a significant content of unsaturated fatty acyl residues, resulting in a high sensitivity against oxidative stress. This is evidenced by the appearance of lysolipids, for example, lysophosphatidylcholine, which acts like a detergent and is normally present in only very small amounts in biological membranes. The epididymis represents a tubular system comprising three main parts (caput, corpus, and cauda), through which the spermatozoa are consecutively transported undergoing distinct maturation stages. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we established three striking differences in the lipid composition of murine spermatozoa from the different epididymal regions: in comparison to the caput sperm, sperm from the cauda are characterized by (1) a higher degree of unsaturation (PC 18:0/22:5 and 18:0/22:6 vs. 18:0/20:4 and 18:0/18:1), (2) an enhanced plasmalogen content, and (3) an enhanced content of lysolipids. These changes are likely to be of physiological relevance and potentially useful as diagnostic markers of sperm maturation and acquisition of motility.

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic "fingerprints" are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.