In Vitro Evaluation of the Permeability of Different Resorbable Xenogeneic Membranes after Collagenolytic Degradation

In this in vitro study, we compare the penetration of cells through different resorbable collagen membranes, which were collagenolytically degraded over different time periods. Three different resorbable collagen membranes were evaluated, including two non-cross-linked (NCL) membranes-namely, a porcine (NCL-P) and an equine (NCL-E) membrane-and an enzymatically cross-linked porcine (ECL-B) membrane. A special two-chamber model was fabricated, allowing for the placement of separating membranes, and a non-porous polyester membrane was used as a negative control (C), in order to verify the impermeability of the experimental chamber device. Round membrane samples with a diameter of 16 mm were fabricated. Eighteen membranes of each type were punched and placed on polyethylene nets as carriers. The membranes were then biodegraded-each on its carrier-in 12-well polystyrene plates: three samples of each membrane type were degraded for 1.5, 3, 6, or 12 h in 2 mL of a buffered collagenase solution, at 37 °C. For control purposes, three samples of each membrane type were not degraded, but only immersed in buffer solution for 1.5, 3, 6, or 12 h, at 37 °C. Another three samples of each type of membrane were degraded until complete dissolution, in order to determine the full hydroxyproline content for comparison. Liquid-preserved boar semen (containing at least 120 million sperm cells per milliliter) was used to test the cell occlusivity of the degraded membranes. At baseline and initial degradation, all tested membranes were tight, and no penetration was observed with up to 30 min of incubation time (results not shown). After 1.5 h, cells were partially capable of penetrating the NCL-E membrane only. One sample showed leakage, with a sperm volume of 1.7 million cells/mL over all samples. No penetration occurred in the test, NCL-P, and ECL-B groups. After a degradation time of 3 h, the NCL-P and ECL-B membranes remained occlusive to cells. All the membranes and measurements indicated leakage in the NCL-E group. After 6 h, four NCL-P measurements showed the first signs of cell penetration, as boar spermatozoa were detectable in the lower chamber (64 million cells/mL). The ECL-B membranes remained completely cell occlusive. After 12 h, four NCL-P measurements were cell penetration positive (14.6 million cells/mL), while the ECL-B group remained tight and showed no cell penetration. As the findings of our study are well in accordance with the results of several previous animal studies, it can be concluded that the surrogate model is capable of performing rapid and cheap screening of cell occlusivity for different collagen membranes in a very standardized manner. In particular, claims of long degradation resistance can be easily proven and compared. As the boar spermatozoa used in the present report had a size of 9 × 5 μm, smaller bacteria are probably also able to penetrate the leaking membranes; in this regard, our proposed study set-up may provide valuable information, although it must be acknowledged that sperm cells show active mobility and do not only translocate by growth

Effects of nutrition on sexual development of bulls

In the last decades a series of attempts have been made to improve reproductive performance of bulls via optimizing nutrition. Although an increase in energy uptake during the post-weaning period of calves led to a faster growing rate, it had no positive effects on sexual development. In contrast, a high-nutrition diet during the prepubertal period reduced the age at puberty of the bulls and increased the size/weight of the testis and the epididymal sperm reserves. This faster sexual development was associated with an increased transient LH peak, which seemed to be mediated by an increase in serum IGF-I concentrations. However, the exact mechanisms responsible for the interaction between nutrition and the subsequent development of calves are still not clear. Sexual development of bull calves depends not only on nutrition of the calves after birth but also on the feed intake of their mothers during pregnancy. A high-nutrition diet fed to the mother during the first trimester has negative effects on the reproductive performance of their offspring. In summary, growth, health and reproductive performance can be improved by nutrition, but further studies are necessary to obtain a better understanding about the mechanisms responsible for this phenomenon

Impact of nutritional programming on the growth, health, and sexual development of bull calves

The growth, health, and reproductive performance of bull calves are important prerequisites for a successful cattle breeding program. Therefore, several attempts have been made to improve these parameters via nutritional programming. Although an increase in energy uptake during the postweaning period (7-8 mo of age) of the calves leads to a faster growing rate, it has no positive effects on sexual development. In contrast, a high-nutrition diet during the prepubertal period (8-20 wk of age) reduced the age at puberty of the bulls and increased the size and/or weight of the testis and the epididymal sperm reserves. This faster sexual development is associated with an increased transient LH peak, which seems to be mediated by an increase in serum IGF-I concentrations. However, the exact mechanisms responsible for the interaction between nutrition and the subsequent development of the calves are not clear. The sexual development of bull calves depends not only on the nutrition of the calves after birth but also on the feed intake of their mothers during pregnancy. In contrast to the effects of the feed intake of the bull calves, a high-nutrition diet fed to the mother during the first trimester has negative effects on the reproductive performance of their offspring. In conclusion, it has been clearly demonstrated that growth, health, and reproductive performance can be improved by nutritional programming, but further studies are necessary to obtain a better understanding about the mechanisms responsible for this phenomenon

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 0.05). 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

Autosomal recessive loci contribute significantly to quantitative variation of male fertility in a dairy cattle population

Background: Cattle are ideally suited to investigate the genetics of male fertility. Semen from individual bulls is used for thousands of artificial inseminations for which the fertilization success is monitored. Results from the breeding soundness examination and repeated observations of semen quality complement the fertility evaluation for each bull. Resultat: In a cohort of 3881 Brown Swiss bulls that had genotypes at 683,609 SNPs, we reveal four novel recessive QTL for male fertility on BTA1, 18, 25, and 26 using haplotype-based association testing. A QTL for bull fertility on BTA1 is also associated with sperm head shape anomalies. All other QTL are not associated with any of the semen quality traits investigated. We perform complementary fine-mapping approaches using publicly available transcriptomes as well as whole-genome sequencing data of 125 Brown Swiss bulls to reveal candidate causal variants. We show that missense or nonsense variants in SPATA16, VWA3A, ENSBTAG00000006717 and ENSBTAG00000019919 are in linkage disequilibrium with the QTL. Using whole-genome sequence data, we detect strong association (P = 4.83 × 10$^{− 12}$) of a missense variant (p.Ile193Met) in SPATA16 with male fertility. However, non-coding variants exhibit stronger association at all QTL suggesting that variants in regulatory regions contribute to variation in bull fertility. Conclusion: Our findings in a dairy cattle population provide evidence that recessive variants may contribute substantially to quantitative variation in male fertility in mammals. Detecting causal variants that underpin variation in male fertility remains difficult because the most strongly associated variants reside in poorly annotated non-coding regions

Development of a flow cytometric assay to assess the bacterial count in boar semen

The aim of the study was to develop a new flow cytometric assay for the determination of the bacterial count in commercially processed boar semen. In total 224 fresh boar semen samples collected at an AI-station were analyzed. The number of total viable counts (TVC) was determined by using flow cytometry after staining with SYBR Green I and Propidium Iodide (PI). In the first part of the study 111 fresh boar semen samples were spiked with pure cultures of defined numbers of bacteria commonly detected in boar ejaculates and analyzed by flow cytometry. In the second part, 113 fresh semen samples were assessed on the day of collection through flow cytometry and the Most Probable Number (MPN) method, as the standard bacteriological method. The first part of the study showed a strong correlation between the detected and expected numbers (r = 0.96; P < 0.001), while in the second part of the study the TVC determined by flow cytometry and by the MPN method correlated only moderately (r = 0.28; P < 0.01; median MPN: 24,000 ± MAD 21,600 bacteria/mL; median flow cytometry: 24,426 ± MAD 15,610 bacteria/mL). In summary flow cytometry is a fast alternative to the classical culture technique to determine highly contaminated boar ejaculates. The developed flow cytometric protocol enables one to enumerate the viable bacteria within fresh boar ejaculates without requiring numerous treatment steps, and thus offering the possibility of an on-line use in AI-centers

Multicolor flow cytometric analysis of cryopreserved bovine sperm: a tool for the evaluation of bull fertility

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI

Sonographische Trächtigkeitsuntersuchung und Bestimmung von Anzahl und Alter der Feten beim Schaf

The aim of the present study was to evaluate accuracy of transabdominal ultrasonography for pregnancy diagnosis, determination of fetal number and estimation of gestational age in ewes. A total of 1068 ewes from 8 different flocks (Swiss White Alpine n=379, Swiss Black- Brown Mountain n=189, Oxford-type n=164, Ostfriesian n=154, Texel n=104, Lacaune × Osfriesian crossbred n=78) was investigated using an Ovi-ScanTM ultrasound scanner with a 3.5 MHz 170° mechanical axial probe (BCF Technology Ltd., Bellshill, Great Britain). Scanning was performed transabdominally at the right inguinal region in ewes restrained in standing position. Sonographic findings were compared with lambing data recorded by the farmers. Included in the analysis were all ewes that, at the time of examination, were not pregnant and those that were pregnant between 26 and 110 days with a known lambing date and number of lambs born (n=882). The sensitivity of pregnancy diagnosis was 97.8% and the specificity 97.6% (overall accuracy 97.8%, n=853). Sensitivity and specificity for discrimination between single and multiple pregnancies was 95.8% and 90.5% (overall accuracy 93.9%, n=752), respectively, when multiples were defined as positive. Discrimination between twins and triplets reached a sensitivity of 86.0% and a specificity of 99.5% (overall accuracy 98.0%, n=458) when triplets were defined as positive. Considering a gestation period of 150 days, the mean deviation of the estimated to the effective day of gestation at examination was 5.6±5.0 days (n=781). The correlation between estimated and true gestational age was very high (r=0.936, P<0.0001). In conclusion, a highly accurate and efficient diagnosis of pregnancy with reliable estimation of fetal number and age is possible using an Ovi- ScanTM ultrasound scanner by an experienced examiner

Analysis of the Pathogenetic Basis for Shedding and Transmission of Ovine Gamma Herpesvirus 2

Ovine herpesvirus 2 (OvHV-2), a member of the viral subfamily Gammaherpesvirinae, shares numerous similarities with human herpesvirus 8 (HHV-8). Both viruses are apathogenic in their healthy original host, may cause lymphoprolipherative diseases, cannot routinely be propagated in cell culture, and may be sexually transmitted. However, the pathways of sexual transmission of these viruses, as well as the underlying pathogenetic dynamics, are not well understood. Organs from naturally OvHV-2-infected, as well as OvHV-2-free, sheep were quantitatively analyzed for OvHV-2 by the DNA amplification techniques. The dynamics of OvHV-2 multiplication and excretion were monitored after experimental infections and, most importantly, subsequent to vasectomy. The OvHV-2 DNA load in various tissues and internal organs was not merely reflecting the viral DNA load in the bloodstream, which suggested compartmentalization of OvHV-2. Moreover, OvHV-2 DNA was detected at several portals for virus shedding, i.e., the respiratory, alimentary, and urogenital tracts. Transient OvHV-2 excretion was detected in ejaculates of experimentally infected rams. Upon vasectomy, OvHV-2 DNA reappeared in the ejaculatory plasma, but the titers did not decline after reaching a peak. Spiking and fractionation experiments revealed an inhibitory activity, associated with the spermatozoa, which was able to suppress detection of viral DNA but which was no longer present in samples from vasectomized animals. Therefore, epidemiological studies on viruses that may be transmitted by the ejaculatory pathway and for whose tracing nucleic acid amplification methods are used, i.e., OvHV-2, HHV-8, and the human immunodeficiency virus, should include vasectomized males

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing-thawing