Relationship between pubertal testicular ultrasonographic evaluation and future reproductive performance potential in Piétrain boars.

New ways of predicting sperm quality and output performance in young artificial insemination (AI) boars are important for breeding companies to ensure that the pubertal boars delivered to the AI studs have a high chance of meeting minimum quality standards to be used for insemination and therewith dissemination of desirable characteristics. The aim of the current study was to characterize the testicular development of 218 pubertal Piétrain boars (Line 408, Pig Improvement Company) to identify traits with predictable characteristics relative to their sperm quality as an adult AI boar. Scrotum, testes and epididymis were examined ultrasonographically at day (d) 100 (on-test) and 170 (off-test) followed by a computer-assisted grayscale analysis (GSA). Over the test period, paired testicular volume increased 7.3-fold from 22.7 ± 10.8 cm3 to 166.6 ± 62.2 cm3. The right testis was significantly (P = 0.014) larger than the left one at the off-test. Based on the sperm quality (ejaculate volume, sperm concentration, total sperm number, morphologically abnormal sperm and total sperm motility at day 3 of semen storage), 82.11% (n = 179) of the boars were classified as "productive" boars. These boars had a significantly (P = 0.039) larger paired testicular volume than "non-productive" boars (45.9 ± 19.9 cm3vs. 38.5 ± 12.6 cm3) at the on-test. For the right testis at on-test, significant differences for the standard deviation of mean gray value (P = 0.022), area under the curve (P = 0.004) and mean gradient value (GRAD, P = 0.030) regarding the future sperm production capacity (SPC) were shown. At off-test, there was a significant difference for minimum gray value (MIN GV, P = 0.003) and mean gray value (P = 0.001) related to SPC. To find SPC related cut-off values for GSA data, a two segmental non-linear regression analysis was carried out indicating breakpoints for GRAD ≥12 and MIN GV ≥ 40 for boars with low SPC. Off-test boars with MIN GV ≥ 40 showed a 2.4 higher risk to display low SPC (Odds ratio = 2.4 [1.1, 5.4]; P = 0.024). The results may enable breeding companies to include new sperm quality associated traits in their boar testing and selection programs.

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.

Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation.

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 µL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13-97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < -0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = -0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Effects of sodium pyruvate on viability, synthesis of reactive oxygen species, lipid peroxidation and DNA integrity of cryopreserved bovine sperm.

The aim of this study was to examine effects of sodium pyruvate on viability as well as on synthesis of reactive oxygen species (ROS), lipid peroxidation and DNA integrity of cryopreserved bovine sperm. In each of 23 Simmental AI bulls three ejaculates were collected. In a split sample design ejaculates were diluted by using Triladyl® extender without and with the addition of 5mM sodium pyruvate. Both aliquots were equilibrated for 24h before freezing. Frozen sperm samples were thawed, and examined immediately after thawing (0h) as well as after 3, 6, 12, and 24h incubation at 37°C. The percentages of rapidly motile sperm (RMS), plasma membrane and acrosome intact sperm (PMAI), sperm with a high mitochondrial membrane potential (HMMP), amounts of ROS synthesis (dichlorofluorescein-diacetate (DCFH), CellROX Deep Red Reagent® probe (CellROX)) and lipid peroxidation of sperm (LPO) and percentage of sperm with a high degree of DNA fragmentation (%DFI) were determined. Overall, sperm diluted with the extender containing sodium pyruvate showed higher levels of RMS, PMAI and HMMP, CellROX and lower %DFI values (P<0.001) compared to sperm frozen in the extender without sodium pyruvate. However, there was no effect (P>0.05) of sodium pyruvate on LPO and DCFH. The results of this study show that the addition of sodium pyruvate to the semen extender improved the viability as well as DNA integrity of cryopreserved sperm and did not affect their lipid peroxidation, although it increased the synthesis of some ROS.

Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen.

This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P < 0.01), and also the extender affected all parameters except HMMP (P < 0.05). After thawing, all parameters except HMMP immediately after thawing were influenced by the equilibration period (P < 0.001), whereas all parameters except % DFI immediately after thawing were influenced by the extender (P < 0.001). The changes of semen characteristics during 3 hours of incubation were also dependent on the equilibration time and the extender used in all parameters (P < 0.01). In the field study, semen of nine bulls was collected thrice weekly, processed using Triladyl egg yolk extender, and frozen in 0.25 mL straws with 15 × 106 spermatozoa per straw. In total, the nonreturn rates on Day 90 after insemination (NRR90) of 263,816 inseminations in two periods were evaluated. Whereas semen collected on Mondays and Wednesdays was equilibrated for 24 hours in both periods, semen collected on Fridays was equilibrated for 4 hours in period one and equilibrated for 72 hours in period 2. No differences in NRR90 could be found (P > 0.05). In conclusion, extension of the equilibration time from 4 hours to 24-72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field.

Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.

The objective was to examine if there are relationships between alterations in sperm viability, reactive oxygen species (ROS) synthesis, and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 hours at 37 °C, and the other frozen, thawed, and incubated for 24 hours at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 hours of incubation. Progressive motile sperm was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm, sperm with a high mitochondrial membrane potential, sperm showing a high degree of DNA fragmentation (%DFI), and their reactive oxygen species content were assessed with dichlorofluorescein-diacetate, dihydrorhodamine, diaminofluorescein diacetate, and mitochondrial superoxide indicator using flow cytometry. Although all other sperm parameters showed alterations (P < 0.05) during the 24-hour incubation time, %DFI stayed constant (P > 0.05, 0.91 ± 0.23) in nonfrozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, dichlorofluorescein-diacetate-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species.

[Lactational incidences of production diseases in German Fleckvieh cows of six Bavarian dairy farms]. / Laktationsinzidenzen von Produktionskrankheiten bei Fleckviehkühen in sechs bayerischen Milchviehbetrieben.

OBJECTIVE: The aim of this study was to assess lactation incidences of production diseases in German Fleckvieh cows. MATERIAL AND METHODS: Investigations were carried out on six dairy farms (mean milk yield of herds 2008: 7834 ± 708kg milk [mean ± SD]) in Bavaria. All farms kept the cows in free stall barns and fed them a total or partial mixed ration based on grass silage and corn silage. In total, 116 cows and 58 heifers were examined daily for 14 days post partum and treated - if necessary - according to standard protocols. The acquisition of data for diseases in the further lactation was carried out by regular visits to the farm as well as communication with the herd manager and the farm veterinarian. RESULTS: Pluriparous cows suffered more frequently from production diseases (milk fever, retained placenta, clinical ketosis, abomasal displacement, metritis, endometritis, ovarian cysts, mastitis) than primiparous heifers: 33.3% and 46.4% of pluriparous and primiparous cows, respectively, remained clinically healthy, while 24.8% and 30.4%, respectively, suffered from one production disease during the first 2 weeks of lactation; more than one production disease was diagnosed in 41.9% and 23.2% of pluriparous and primiparous cows, respectively. The lactation incidences of production diseases varied considerably among pluriparous cows of the six farms: retained placenta 16.8 ± 13.2%, milk fever 15.1 ± 7.0%, clinical ketosis 16.8 ± 12.4%, metritis 3.8 ± 3.1%, abomasal displacement 1.1% (median 0.0; 0.0/0.0%), endometritis 11.7 ± 7.0%. Mastitis affected 56.0 ± 7.4% of the pluriparous cows, which experienced 1.7 mastitis episodes on average. At least one follicular cyst was diagnosed among 28.4 ± 8.6% of the cows. Lameness affected 18.5 ± 13.5% of pluriparous cows and heifers during the first 2 weeks of lactation. CONCLUSION: The lactation incidences of production diseases did not significantly differ from reference values reported for Holstein Friesian cows except the lower incidence of LDA among German Fleckvieh cows. The results indicate that the farm management affected lactation incidences of production diseases to a greater degree than additional factors, such as the breed of the cows.

Liposomes for cryopreservation of bovine sperm.

In this study, the effect of various unilamellar liposomes on cryopreservation of bovine spermatozoa has been investigated. Liposomes were composed of saturated lipids with various acyl chain lengths: DSPC (18:0), DPPC (16:0), DMPC (14:0), or DLPC (12:0). Alternatively, liposomes were prepared using unsaturated egg phosphatidylcholine (EPC) or DOPC (18:1, neutral), alone or in combination with lipids with various head groups: DOPS (negatively charged), DOPG (negatively charged), and DOPE (neutral). Fourier transform infrared spectroscopy studies showed that bovine sperm membranes display a gradual phase transition from 10 to 24 (o)C. Phase transition temperatures of the liposomes varied from -20 to +53 (o)C. Sperm was incubated in the presence of liposomes for either 6 or 24 h at 4 °C prior to freezing. Postfreeze survival rates were determined based on the percentage of progressively motile cells as well as the percentage of acrosome- and plasma membrane-intact cells. With DOPC liposomes a postthaw progressive motility of 43% was obtained compared with 59% using standard egg yolk freezing extender. Postthaw progressive motility increased up to 52% using DOPC:DOPG (9:1) liposomes, whereas DOPC:DOPS or DOPC:DOPE liposomes did not increase survival compared with DOPC liposomes. Among the saturated lipids, only DMPC was found to increase cryosurvival, up to 44% based on progressive motility. DLPC liposomes caused a complete loss in cell viability, already prior to freezing, whereas DPPC and DSPC liposomes neither positively nor negatively affected cryosurvival. Taken together, the higher postthaw survival obtained with DOPC:DOPG liposomes as compared with DOPC liposomes can likely be attributed to increased liposome-sperm interactions between the charged phosphatidylglycerol groups and charged regions in the sperm membranes. Interestingly, the lipid phase state of the liposomes during preincubation is not the decisive factor for their cryoprotective action.

[Field studies of the effect of sperm motility and morphology on the fertility of boars used for insemination]. / Spermienmotilität und -morphologie in ihrer Auswirkung auf die Fruchtbarkeit von Besamungsebern in Feldversuchen.

Based on 2127 first services in a field trial, the influence of sperm motility and morphology on the fertility of ten AI boars was investigated using semen stored for three and five days. Depending on sperm morphology the farrowing rate differed by up to 31% and the litter size differed by up to 3.4 piglets. Sperm motility and morphology are useful parameters in selecting sires for AI, especially in the case of inseminations with semen stored long-term.

Influence of chlorhexidine on seminal patterns in stallions.

Ejaculates were collected at 3-day intervals before, during and after a washing procedure with chlorhexidine (2%). Semen motility and pathology were determined before and after deep-freezing. Blood samples were taken before and within 1 h after washing procedures and then extracted in ether. This was followed by HPL chromatography. Chlorhexidine concentrations in blood and seminal plasma were distinctly higher in the treated stallions than in control groups. Concentrations in the control groups were below the detection limit of the column. Significant correlations between decreasing semen quality (% immotile cells, % morphological aberrations) and chlorhexidine treatment were found. A change of the present requirements for imports into North America should be considered to improve the semen quality of deep-frozen ejaculates.